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THE 
ACTINOMYCETES 
VOLUME TI 


Ferdinand Cohn (1828-1898), who was the first to observe and describe an actino- 
mycete (1875), under the name Streptothriz Foerstert. 


THE ACTINOMYCETES 
Volt 
CUA ssh CATLON 2 DENY EEDCA TON AN D 


DESGHRIPTIONS OF GCENERA AND SPECIES 


by 


Selman A. Waksman 


BALTIMORE 
iE Web ieh FAV Sivek WEE KILNS GCOMPAN Y 
1961 


THE ACTINOMYCETES 


Vou. II: CLASSIFICATION, IDENTIFICATION AND 
DESCRIPTIONS OF GENERA AND SPECIES 


Copyright ©, 1961 
The Williams & Wilkins Company 


Made in the United States of America 


Library of Congress 
Catalog Card Number 
59-9962 


Composed and printed at the 
WAVERLY PRESS, INC. 
Baltimore 2, Md., U.S.A. 


PREFACE 


In 1922, Professor D. H. Bergey of the University of Pennsylvania wrote to 
me that he and the Committee on Characterization and Classification of the 
Society of American Bacteriologists were in the process of preparing a ‘‘Manual 
of Determinative Bacteriology’’; he asked whether I would be willing to under- 
take the preparation for that volume of a section dealing with the actinomycetes. 

This group of organisms had occupied my attention for the previous several 
years, and alone (1919) and with Roland Curtis (1916), I had deseribed a num- 
ber of new species; yet I hesitated to accept this assignment. There were several 
important reasons for this hesitation: (a) I was not at all sure that the descrip- 
tions of actinomycetes so far published provided sufficient information for the 
accurate identification of most of the species recorded in the literature; (b) only 
four years previously, | had been warned by the dean of American cryptogamic 
botanists, Roland Thaxter, not to make further descriptions of new species based 
solely or largely upon cultural and biochemical properties; and finally (¢) I was 
not even certain at that time whether the actinomycetes should be included 
with the bacteria. 

I told Professor Bergey all this and suggested that it would be better to wait 
a few years until more detailed information was obtained concerning this group 
of microorganisms, especially with regard to their morphological and biochemical 
properties, before an attempt was made to codify them. I received a curt and 
somewhat sarcastic reply that if I would not, for one reason or another, under- 
take this task, he would have to do it himself. My immediate answer was, ‘‘I 
will do it.”? The best that I could accomplish at that time was to use cultural and 
biochemical characteristics as a major basis for the classification of the actinomy- 
cetes and for the characterization of the known species. 

Since then, or for more than a third of a century and for seven consecutive edi- 
tions of ““Bergey’s Manual,” I have been largely responsible for the preparation 
of the descriptions of the actinomycetes. I have not, however, always had the 
final word in organization of the material for all the various editions. Alone 
(1940), and together with Professor A. T. Henrici of the University of Minnesota 
(1943), I proposed two systems for classification of the actinomycetes, the second 
of which consisted of a thorough revision of the group and its separation into four 
genera. The most significant change in this revision was the proposal, in 1943, of 
the new generic name, Streptomyces. This second system has been the basis for 
the organization of the material in the last two editions of Bergey’s Manual. 

In presenting this volume, I am now certain of one thing, namely, that the 
place of the actinomycetes is definitely among the bacteria and not among the 
fungi. Ample evidence of this belief has been presented in Volume I of this trea- 
tise. Unfortunately, the first reason for my hesitancy in 1922, I believe, remains 
valid; the accuracy of the information available for species identification is still 


Ve 


vl THE ACTINOMYCETES, Vol. II 


open to question. The chief reason for this uncertainty is that although much 
knowledge has since accumulated, especially during the last 20 years when many 
Streptomyces species became known as antibiotic-producing organisms, taxonomic 
work was largely neglected except by a few dedicated investigators. Recently, 
however, several important contributions (Hesseltine et al., 1954; Flaig and 
Kutzner, 1954; Kutzner, 1956; Waksman, 1957; Ettlinger et al., 1958; Pridham, 
1959) to this subject have appeared. A survey of the recent literature shows that 
morphological characters are tending to replace physiological and cultural proper- 
ties as the leading criteria in species characterization. It may be said that we are 
now in a transitional stage in which our ideas are changing, not only concerning 
the usefulness of criteria for species differentiation, but also with regard to the 
species concept. Since a classification of a group of living organisms is always only 
‘preliminary,’ based upon the current knowledge of these organisms, I believe 
that, in summarizing the subject at present, and in trying to combine the older 
and newer ideas, I have presented useful criteria for species differentiation and 
an outline of species concept for the genera Actinomyces, Nocardia, Streptomyces, 
Micromonospora, and certain others. 

The rapidly accumulating information about the separation of some of the 
genera into distinct groups or sections, the recent introduction of several new 
genera, and the description of numerous new species, all necessitated a complete 
recasting of the material presented in the last edition of Bergey’s Manual and in 
other treatises. This volume is largely the result. An attempt has been made to 
bring together in this volume all the information required for the identification 
of newly isolated cultures of actinomycetes. All descriptions and names for which 
insufficient data have been provided, especially when no reproducible media have 
been employed, have been placed in a separate chapter as ‘‘incompletely de- 
scribed.”? Descriptions in which excessive and often confusing information has 
been presented, have been abbreviated to fit a certain ‘‘standard.”’ Often, this 
standard has turned out to be a Procrustean bed. I beg forgiveness, both from 
the ‘‘reader” and from the preservers of the Code (International Code of Nomen- 
clature of Bacteria and Viruses). My sole apology is that it is my sincere hope 
that it would serve the purpose. 

The author wishes to acknowledge his sincere indebtedness to Dr. Norvel M. 
McClung of the University of Georgia, to Dr. R. E. Buchanan of Iowa State 
University, and to Dr. Ruth E. Gordon and Dr. Hubert A. Lechevalher of this 
Institute, for reading individual chapters and for making valuable suggestions; 
to Dr. Hans J. Kutzner of this Institute and Dr. Thomas G. Pridham of the 
Northern Regional Research Laboratory, for reading the major portions of this 
volume and for suggesting numerous corrections and modifications; to Miss Alma 
Dietz of the Upjohn Company, Dr. Edward J. Backus of the Lederle Labora- 
tories, and all others who kindly supplied photographs; to Mrs. Herminie B. 
Kitchen for editorial work, and to Mr. Robert A. Day for assistance in the prep- 
aration of the various illustrations and for reading the entire manuscript, 

Selman A. Waksman 


INTRODUCTORY 


This volume deals exclusively with the well recognized genera of the actino- 
mycetes. No consideration is given here to the various closely related genera that 
are often included in the order Actinomycetales, notably the genus Mycobacterium 
Lehmann and Neumann, 1896. 

The actinomycetes comprise three families, which are further subdivided into 
LO genera. 


A. Spores formed, but not in sporangia. 
I. Vegetative mycelium fragmenting into bacillary or coccoid elements. 
Family I. Actinomycetaceae Buchanan. 
1. Anaerobic or microaerophilic, nonacid-fast. 
Ll. Actinomyces Harz 
2. Aerobic, partially acid-fast or nonacid-fast. 
2. Nocardia Trevisan 
II. Vegetative mycelium nonseptate, not fragmenting into bacillary or coc- 
coid elements. 
Family II. Streptomycetaceae Waksman and Henrici. 
1. Aerial mycelium produced. 
a. Spores formed in chains. 
3. Streptomyces Waksman and Henrici 
b. Spores formed singly. 
4. Thermoactinomyces Tsiklinsky 
c. Spores occurring in pairs or in chains. 
a'. Mesophilic forms, in pairs. 
5. Waksmania Lechevalier and Lechevalier 
b!. Thermophilic forms, in pairs or in chains. 
6. Thermopolyspora Henssen 
2. Aerial mycelium not produced. 
a. Spores occurring singly on short sporophores. 
al. Mesophilic forms. 
7. Micromonospora Orskov 
b!. Thermophilic forms. 
8. Thermomonospora Henssen 
B. Spores occurring in sporangia. 
Family III. Actinoplanaceae Couch 
I. Aerial mycelium usually not formed, coiled conidial chains lacking, spor- 
anglospores motile. 
9. Actinoplanes Couch 
II. Aerial mycelium abundant, coiled conidial chains as well as sporangia 
formed in some species, sporangiospores nonmotile. 
10. Streptosporangium Couch 


vil 


vill THE ACTINOMYCETES, Vol. IT 


Certain other genera, recently suggested, have been given tentative considera- 
tion. 

These genera comprise about 350 species. In addition to these, a large number 
of other species are listed as ‘incompletely described.” 


TABLE OF CONTENTS 


The Actinomycetes 


VoLuME II 


CLASSIFICATION, IDENTIFICATION, AND DESCRIPTION 
OF GENERA AND SPECIES 


PTE RC Cah Oreo Se Ba wig es. RPSL Aci: Ste Pant ess A Bn sk a a I Ae tne V 
MNT OCCU OTS chance co o.8 Soi a Pe te, a wna Neer Gik-oth a MeL eee vil 

1. The Species Concept in Relation to the Actinomycetes.............. | 
Deal CUE E MUSA CLEMOMUCES 2:0 See or Pale a heaas ves seas Foe oes eee tad ee exe 12 
“ah IU OEM (ey NILOTSENA'A(O170 1410 110 NEA ee Pe oe Be a oes ot mE cm ne 21 
4. Characterization of Streptomyces Species.............. 02.0000 eee 61 

5. Systems of Classification and Identification of Groups and Species of the 

GSMS HSU DLONUNCCG tice ole gts oie lois ijt Fe Boas e SeOPO ta Ae ne oe oR 82 

6. Series and Species of the Genus Streptomyces... ........0.0...0.000-. LS 
fe Classiication ob wieplomiyces SPeCles.. - 2.6 ce. bebe ecw ove see nies 152 
S. Deseriptionror Species of Streptomyces... o.oo wee ne 165 
Omiihe: Genus Mecromonospordy, les ele. Slee els ca da See lasagne saan 293 
LOMeithesGenusaw aksmanica (Microbtspora)): co ..08 2 fee le hs oso ed Ss 298 
ea hiermopnilicy Actinomycetes. ..tsS sob. ae Oe bs bo awlegan eee ba bs 300 
WAAC HINO DIAM ACCACUe +. oes teas es es Be aoe 2 eee 310 
13. Incompletely Described Species of Actinomycetes................... 315 


Appendix I. Color Designations for Describing Actinomycetes (Lin- 


GLEMDEII) Eee Ee UPA AI Mente sae. Slee CEs EO OS oat te eee 327 
Appendix II. Certain Important Media for the Study of Actinomycetes 328 
| 572) (EN 2) CET ARN Oa mane ae Maen Se rd OS eee ae ee) AE 335 
InICexXAG MOTE AIMISMIS SSeS eo ENA Ee cl Meee 347 
Crenerall Sinden... Mle tice, at teas: syns Neen e cae Uy cake mute clog CR Lt een eee 360 


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Chapter |! 


The Species Concept in Relation to 


the Actinomycetes 


Systematic Position of the Actinomy- 
cetes 

In the preface to this volume, the state- 
ment was made that ‘I am now certain of 
one thing, namely, that the place of the 
actinomycetes is definitely among the bac- 
teria and not among the fungi. Ample evi- 
dence of this belief has been presented in 
Volume I of this Nevertheless, 
some reiteration is warranted at this point. 

The taxonomic position of the 
mycetes, notably their relationship to the 
bacteria, on the one hand, and to the fungi, 
on the other, has been one of the most de- 
batable questions in microbiology. The size 
(width of thallus) and staining properties of 
the actinomycetes have usually placed them 
with the bacteria. Their branching and man- 
ner of sporulation have suggested their rela- 
tionship to the fungi. Still other properties of 


treatise.”’ 


actino- 


actinomycetes seemed to warrant their con- 
sideration as a transition group between the 
bacteria and the fungi. 

tecent evidence seems to point definitely 
to the fact that the actinomycetes are more 
closely related to the bacteria: 

1. Some of the actinomycetes, such as 
species of Actinomyces and Nocardia, are 
closely related to true bacteria, notably spe- 
cies of Lactobacillus and Corynebacterium. 

2. Neither 
have been shown to contain true nuclei; they 
both contain only chromatin granules dis- 
tributed through the hyphae or the cells. 


actinomycetes nor bacteria 


3. The diameter of actinomycete my- 
celium and spores is similar to that of bac- 
teria. Actinomycetes also, as a rule, lack 
septa. 

4. Actinomycetes are subject to attack by 


phages just as bacteria are; filamentous 
fungi are not. 
5. Actinomycetes are usually sensitive 


(allowing for strain variability) to antibiotics 
that are active upon bacteria; they are usu- 
ally resistant to those antibiotics, like the 
polyenes, that are active upon fungi but not 
upon bacteria. 

6. Chitin is absent from the cell substance 
of actinomycetes as well as from bacterial 
cells, but is present in fungus mycelium and 
In their lack of cellulose, actino- 
mycetes are also similar to most bacteria and 
unlike fungi. Avery and Blank (1954) con- 
cluded that “from the chemical point of view 
Actinomycetales have nothing in common 
with the true fungi, but rather with the bac- 
Cummins and Harris (1958) 
even further by suggesting that the order 


spores. 


teria.”’ went 
Actinomycetales be abolished altogether and 
that the families of the actinomycetes be 
included in the Hubacteriales. 

7. Like bacteria, but unlike most fungi, 
actinomycetes as a rule are sensitive to 
an acid reaction of the medium. 

8. The close relationship of the actino- 
mycetes to the bacteria is also evident from 
the work of Couch (1954), who found that 
certain J/icromonospora-like forms resemble 


2 THE ACTINOMYCETES, Vol. II 


those of bacteria. Couch emphasized the re- 
semblance of the mycelium and sporangia of 
Actinoplanes to those of the chytrids; he con- 
cluded that this genus may represent a con- 
necting link between the bacteria and the 
lower fungi. 


The Generic Problem with Actinomy- 
cetes 

Prior to 1943, several systems of classifi- 
cation of actinomycetes had been proposed. 
In most instances, all the species were in- 
cluded in a single genus, which was fre- 
quently designated by different names. The 
most common of these names were the two 
oldest, Streptothrix and Actinomyces. <Al- 
though occasional efforts had been made to 
separate the actinomycetes into several gen- 
era, such attempts usually failed to receive 
more than passing attention. The work of 
Waksman (1919), @rskov (1923), Jensen 
(1931), and Erikson (1935) finally led Waks- 
manand Henrici to suggest, in 1948, the divi- 
sion of the actinomycetes into four genera. A 
new genus, Streptomyces, was proposed to in- 
clude those forms that are characterized by 
the production of an aerial mycelium with 
catenulate spores. Most of the important an- 
tibiotic-producing organisms subsequently 
have been found to belong to this genus. 

Unfortunately, this generic separation 
brought with it a number of new problems, 
which can be briefly summarized as follows: 

1. There is considerable overlapping 
among the different genera, notably between 
certain forms of Streptomyces that have lost 
the capacity to produce aerial mycelium and 
species of Nocardia, as brought out in a re- 
cent paper by Gordon and Smith (1955); 
there is also overlapping between certain 
nocardiae and mycobacteria. 

2. The formation by species of Strepto- 
myces and by certain forms of Nocardia of 
two different types of mycelium, substrate 
and aerial, and the influence of previous con- 
ditions of cultivation upon the growth and 


biochemical activities of these organisms 
served to confound the existing confusion. 

The nomenclatural status of the genera of 
Actinomycetales has recently been discussed 
by Lessel (1960). 

Lechevalier et al. (1961) described a new 
genus Micropolyspora (type species M. bre- 
vicatena), an organism that fragments like 
the members of the family A ctinomycetaceae 
and sporulates like a member of the Strepto- 
mycetaceae, by forming chains of conidia on 
aerial hyphae; it also forms chains of conidia 
on the substrate mycelium. These authors 
suggested that the family Streptomycetaceae 
be dropped and the family A cténomycetaceae 
be enlarged to include the genera Actinomy- 
ces, Muicromonospora, Thermoactinomyces, 
Waksmania, Micropolyspora, Nocardia, and 
Streptomyces. 


What Is a Microbial Species? 


In the study of the taxonomy of any group 
of living organisms, including microorgan- 
isms, one is faced sooner or later with the 
problem of defining what is meant by a 
species. With microorganisms, in usual prac- 
tice, a microbial culture is designated by 
a name, sometimes qualified with a strain 
number; its morphological and cultural 
properties, and frequently its ecological and 
etiological characteristics, are described suf- 
ficiently so that anyone who finds this or- 
ganism in nature will be able to recognize it 
from the description. If possible, the type 
form of the species is preserved in a type 
culture collection, to aid in the future identi- 
fication of the species. 

Unfortunately, microbial forms and types 
of organisms are not fixed in nature or even 
in culture. Some strains, even those closely 
related to the fixed type, may differ enough 
to raise a question as to their exact or specific 
identity. This frequently leads, often on the 
basis of only minor differences, to the crea- 
tion of new species that are given new epi- 
thets. This is particularly true of those mic- 


SPECIES CONCEPT IN RELATION TO ACTINOMYCETES 


roorganisms, like the actinomycetes, that 
occur abundantly in nature; some of the 
newly isolated cultures may differ greatly 
from the fixed types. The difficulty of estab- 
lishing and recognizing ‘“‘species’”’ under these 
conditions may become particularly perplex- 
ing. Raper (1954) was fully justified in say- 
ing, “It is almost axiomatic that the ease 
with which a species of microorganism can be 
recognized tends to vary inversely with the 
number of isolates available for observation 
and examination.” 

The concept of ‘“‘species”’ first used during 
the seventeenth century gradually came to 
denote the fundamental units of a biological 
classification. These units came to be re- 
garded as fixed or static entities, created by 
nature, which can be grouped into higher 
categories, namely, genera, orders, and 
classes. As the evolutionary theory was grad- 
ually accepted, especially with the develop- 
ment of modern genetics and cytology, the 
concept of ‘‘species” began to undergo a 
change. 

Hucker and Pederson (1931) emphasized 
that the difficulty of dividing lower forms 
into well-defined species has led many to 
question whether these are natural groups 
and whether they can be considered to be 
similar to ‘“‘species’”? among higher forms of 
life. The problem always arises: How much 
difference must exist between two cultures 
of bacteria before we are justified in regard- 
ing them as distinct species? 

Krassilnikov (1938) was very emphatic in 
stating that many investigators, without 
considering the rules of nomenclature pro- 
posed at international congresses, either de- 
scribe the same forms under different names 
or combine various organisms into the same 
species. He said: ‘Even the concept of 
‘species’ is considered differently by various 
workers depending on their individual point 
of view, frequently considering a minor lack 
of correlation of a certain character as suffi- 
cient justification for creating a new species.” 


oe 


ae 


Just as in the case of many groups of true 
bacteria, one of the causes of the chaotic 
state of nomenclature of the actinomycetes 
is the lack of type cultures. It has actually 
been suggested (Skerman, 1949) that even 
the available cultures be completely rede- 
scribed, priorities being based on existing 
names, and those names and descriptions for 
which no type cultures are available be dis- 
sarded. 

In comparing the species concept among 
microbes with that of higher plants and 
animals, Cowan (1956) suggested that con- 
sideration be given to the following aspects: 
(a) whereas larger plants and animals have 
geographical distribution areas, few microbes 
have such particular areas; (b) morphology 
is essential for the separation of species 
among algae, fungi, and protozoa, but. it 
barely distinguishes higher ranks among bac- 
teria; (c) cytology is useful at the generic 
level, but ‘‘at the species level the bacteriol- 
ogist relies more on physiological than on 
morphological differences”; (d) interfertility 
is hardly to be considered as a species char- 
acter, since bacteria and actinomycetes re- 
produce asexually; (e) the introduction of 
certain characters in microbiology not uti- 
lized by botanists and zoologists adds satis- 
factory classification criteria; these include 
“nutritional requirements, metabolic and 
‘atabolic products, antigenic structure and 
pathogenicity.” 

In discussing bacterial classification, 
Sneath (1957) came to the following con- 
clusions: (a) an ideal classification is one 
which has the greatest content of informa- 
tion; (b) over-all similarity is the basic con- 
cept of such an ideal classification, and is 
measured in terms of the number of similar 
features possessed by two organisms; (c) 
every feature should have equal weight; (d) 
the division into taxonomic groups is made 
upon correlated features. 

To avoid the growing confusion from con- 
flicting ideas, Gilmour (1958) suggested sep- 


4 THE ACTINOMYCETES, Vol. II 


aration of the concepts of “nomenclatural 
taxonomy” from those of ‘experimental tax- 
onomy.”’ It is to be remembered that species 
are, after all, convenient ‘“‘artificial creations 
of human imagination” rather than “‘real 
biological entities.”” Gilmour further sug- 
gested that “nomenclatural categories of 
genus, species, variety, etc.” are excellently 
suited for the purpose of ‘‘a broad map of 
the diversity of living things.” It would, 
therefore, be ‘‘a great advantage if they were 
not subject to continued attempts to bring 
them up to date and to redefine them in 
evolutionary terms.” 


Speciation of Actinomycetes Other than 
Streptomycetes 


Kxrassilnikov (1938) wrote, ‘In spite of the 
most extensive literature, we have no definite 
idea concerning the natural systematics of 
the actinomycetes, nor a single opinion of 
their structure and development.’ The re- 
cently accumulated information leads us to 
conclude, however, that we need not be so 
pessimistic. 

According to Pridham (1959), there are 
now known more than 100 genera of actino- 
mycetes and well over 1500 subgeneric names 
and specific, or subspecific, epithets. Some of 
the descriptions of these forms are good, 
others lack essential details, and many are 
worthless. Morphological criteria are be- 
lieved to play an important role in separa- 
tion at the generic level (Fig. 1), with a 
gradual intergradation in complexity of re- 
productive units. The actinomycetes are 
looked upon as a heterogeneous group of 
organisms, ranging from the simple myco- 
coeci and the seemingly more complex no- 
vardiae to the straight or flexuous strepto- 
mycetes and the verticillate forms, and from 
the relatively simple micromonosporae to 
forms such as Waksmania, Actinoplanes, and 
Streptosporangium (the latter two genera pos- 
sibly having some affinities with the chy- 
trids). Some of these organisms have definite 


affinities with true bacteria, others with both 
bacteria and microfungi, and still others with 
phycomycetous fungi. 

This heterogeneity is further emphasized 
by the facts that the actinomycetes contain 
forms that are anaerobic, microaerophilic, or 
aerobic; forms that fragment and those that 
do not; and forms that produce aerial my- 
celium and those that do not. Pridham sug- 
gested that some of the present concepts 
centered around the three genera Actino- 
myces, Nocardia, and Streptomyces be ac- 
cepted. Thus included in the Actinomyces 
would be the anaerobic to microaerophilic 
forms; in the Nocardia, the aerobic types 
that either form no aerial mycelium or pro- 
duce an aerial mycelium that generally has 
no catenulate spores; and in the Streptomy- 
ces, the aerobic forms that generally produce 
satenulate spores. 

Although time and again taxonomists have 
emphasized that an effective system of clas- 
sification should be based upon criteria that 
are expressed in consistently reproducible re- 
sults, this has hardly been applied, at least 
so far as our present knowledge is concerned, 
to the species characterization of actino- 
mycetes. Many ‘‘new species’”’ have been de- 
seribed on the basis of a single difference— 
frequently a quantitative variable—from 
“old species.”? One often wonders what the 
composition of the medium, the conditions of 
growth, and the natural variability observed 
so frequently among duplicate cultures have 
to do with these distinguishing properties. 

The species concept among the actinomy- 
cetes must be considered as the continuity 
between different groups of organisms desig- 
nated as species, with various transitional 
forms bridging the gaps between species. The 
concept of natural classification apples to 
actinomycetes perhaps better than to many 
other bacterial groups: there are the chemi- 
cal approach (chemical composition, pres- 
ence of specific chemical compounds), the 
morphological approach (type of aerial my- 


SPECIES CONCEPT IN RELATION TO ACTINOMYCETES 5) 


Mycococcus 7 


Jensenia l \7 
Polysepta ye Lee 


Mycobacterium ('\ Y 


Actinomyces } fe 


A 
; é 
Nocardia 
Proactinomyces 
Pseudonocardia 
B 


Streptomyces 


Chainia® 


Streptoverticillium* 


Micromonospora 


Thermoactinomyces 


Thermomonospora ee 


Microbispora? 
Thermopolyspora; 


Waoaksmania 


ee 
Actinoplanes yz - wo 
Streptosporangium Af 


Fraure 1. Morphology of the various genera of the Actinomycetales. Of these, only Actinomyces in 


A, Nocardia in B, Streptomyces in C, Micromonospora, Thermoactinomyces, Waksmania. Actinoplanes 
i 4 I q I ) 


and Streptosporangium in D are recognized in this treatise as true actinomycetes; Nocardia and Proac- 


tinomyces are synonyms (Courtesy of T. 
Research Service, U. S. Department of Agriculture). 


G. Pridham of the Northern Regional Laboratory, Agriculture 


6 THE ACTINOMYCETES, Vol. II 


celium, type of sporulation, shape and sur- 
face of spore), and finally the ecological ap- 
proach (anaerobie versus aerobic, pathogenic 
versus nonpathogenic, thermophilic versus 
mesophilic). The idea of a physiological clas- 
sification includes formation of antibiotics 
and of enzymes, utilization of carbon com- 
pounds, and transformation of nitrogenous 
compounds, all of which can supply supple- 
mentary information. 


Speciation of Streptomycetes 


What has been said for the actinomycetes 
as a whole applies particularly to the large, 
heterogeneous, and variable group of organ- 
isms represented in nature by the aerial my- 
celium-producing strains, most of which are 
included at present in the genus Streptomy- 
ces. These organisms are found in the soil in 
the form of hundreds of thousands of spores 
and of bits of mycelium per gram. They are 
also found extensively in manures and in 
composts, in various fresh-water basins, in 
dust, and on food. They are almost entirely 
absent from peat bogs and the sea. 

The actinomycetes belonging to the genus 
Streptomyces have recently come to occupy 
an eminent place because many of them are 
important producers of antibiotics, vitamins, 
and enzymes. 

With the growing economic significance of 
members of the genus, the establishment for 
each species of certain characteristics which 
would be adequate to enable the investigator 
to recognize freshly isolated cultures in well 
defined specific terms becomes of great theo- 
retical and practical importance. 

Following the first descriptions of Cohn 
(1875), very few additional species of the 
aerial mycelium-producing actinomycetes 
were recognized until 1914. This was true in 
spite of the rapidly accumulating literature 
on the occurrence of such actinomycetes in 
the soil and in the causation of plant diseases. 
The designations limited 
largely to the names ‘‘Actinomyces albus”’ 


common were 


and ‘‘Actinomyces chromogenus,” depending 
on the color of the aerial mycelium or the 
formation of soluble, dark pigments in com- 
plex organic media. 

Rossi-Doria (1891) was the first to de- 
scribe an organism, under the name Strepto- 
thrix alba, which was later designated as the 
type of the genus Streptomyces proposed by 
Waksman and Henrici in 1943. The most 
important characteristics of this species are 
its white aerial mycelium and the tendency 
for colonies to form concentric rings of this 
aerial mycelium. Rossi-Doria noted the abil- 
ity of his organisms to grow on numerous 
complex organic substrates. 

Thaxter (1891), who first described an im- 
portant economic species, the causative 
agent of potato scab (which he believed to 
be a fungus, Oospora), was highly critical of 
the efforts to describe ‘‘species”’ largely on 
the basis of cultural properties of the organ- 
isms. In this respect, the actinomycetes do 
not differ from any of the other groups of 
bacteria, where cultural properties and bio- 
chemical reactions have to supplement in- 
sufficient morphological information. Physi- 
ological activities and ecological properties, 
which are the expression of the response of 
organisms to their environment, are too 
numerous and often too variable among ac- 
tinomycetes to justify unlimited confidence. 

Krainsky (1914), Waksman and Curtis 
(1916), and Waksman (1919) emphasized 
the use of synthetic substrates, mn addition 
to organic media. Carbon and nitrogen utili- 
zation tests were employed. Added attention 
was given to micromorphology. Many new 
species were described. Jensen (1930a, 1931) 
and Duché (1934) added various new species, 
the latter investigator stressing the use of 
various combinations of carbohydrates and 
nitrogenous compounds as media ingredients. 

One of the reasons for the limited recog- 
nition of species among the aerial mycelium- 
producing actinomycetes prior to 1914 was 
the fact that protein-rich media were 


SPECIES CONCEPT IN RELATION TO ACTINOMYCETES 


employed for their cultivation. With the in- 
troduction of synthetic media, it became 
definitely established that the aerial myce- 
lium-producing actinomycetes comprise a 
large number of forms, differing greatly in 
their physiological and biochemical proper- 
ties, and to a lesser degree in their morphol- 
ogy. It was also recognized that, if a suffi- 
ciently large number of cultures was isolated 
and examined, many differences would be 
noted suggesting variability of the type spe- 
cies. The concept ‘‘species-groups,”’ with one 
culture as the type species, was suggested. 
Waksman (1919) emphasized, therefore, that 
in spite of variation of individual biochemi- 
cal characteristics of the actinomycetes, 
there are certain well defined properties, no- 
tably morphology, color of aerial mycelium, 
and formation of soluble pigments, that char- 
acterize these organisms, especially when 
grown on standard synthetic media and un- 
der carefully controlled conditions of tem- 
perature and aeration. 

It is easy to pick out a few cultures of 
actinomycetes (or streptomycetes) which 
possess characteristic properties that can be 
recognized as distinct species, and to discard 
all the others. This was actually done by 
Waksman and Curtis in their early (1915- 
1916) classification of actinomycetes, since 
they were faced with such a large number of 
freshly isolated cultures that it was impos- 
sible to consider more than a very small 
number of them. How many others have 
acted likewise it is difficult to say. Should 
the various intermediate strains be consid- 
ered, one might be inclined to regard each 
as a different species, distinct from the 
others in at least one variable property, be 
it morphological, cultural, or biochemical. 
With the examination, in recent years, of 
many thousands of cultures of actinomy- 
cetes for their antibiotic properties, such an 
attitude was frequently reduced to an ab- 
surdity. There are those who contend that 
the insistence on permanent characteristics, 


~I 


preferably a group of them, in describing new 
species, would limit greatly our recognition 
of the growing economic importance of these 
organisms. Then there are those who reason 
that not enough species of actinomycetes 
have so far been described, thus justifying 
random descriptions of many freshly isolated 
strains as new species. 

Even synthetic media did not yield the 
final answer to the species problem of this 
group of organisms. Their cultural proper- 
ties, or growth characteristics in media of 
different chemical composition, properties 
that were at first greatly emphasized, were 
found to be extremely variable. Type cultures 
were shown to change their specific charac- 
teristics when grown in artificial media. 
Saltations and mutations came to play a 
highly important part in changing such prop- 
erties. When morphology was recognized at 
all, it was limited largely to observations on 
the curvature of the sporophores or to the 
size and shape of the spores. Drechsler (1919) 
was the first to make a detailed study of the 
morphology of the actinomycetes that pro- 
duce aerial mycelium. Unfortunately, he 
limited his study to a small number of cul- 
tures; this prevented him from establishing 
the existence of many specific types which 
could have been recognized on the basis not 
only of cultural but also of morphological 
properties. 

It must be regarded as a considerable step 
backward when Lieske (1921) completely 
disregarded the work of Krainsky (1914), 
Waksman and Curtis (1916), Conn (1917), 
and Waksman (1919). He believed that the 
classification of actinomycetes was impos- 
sible, since the properties observed were 
highly variable. His skeptical attitude to- 
ward the question of speciation of actino- 
mycetes was due largely to his use of complex 
media for the growth of these organisms, and 
to a lack of sufficient appreciation of the 
significance of simple media for their char- 
acterization. 


8 THE ACTINOMYCETES, Vol. II 


Burkholder et al. (1954) were led to con- 
clude that the species concepts formulated 
by an individual investigator depend a great 
deal upon the investigator’s personal expe- 
rience, and whether he is a ‘‘splitter’’ or a 
‘“Jumper.”’ They suggested further that mi- 
crobial species should be characterized by 
multiple, readily recognizable, and reason- 
ably stable properties; the history of the 
cultures and the nature of the medium in 
which they are growing are of prime im- 
portance. 

With the genus Streptomyces gaining con- 
siderable economic importance, the creation 
of many new species based upon biochemical 
properties, notably formation of antibiotics, 
resulted in much confusion in the recognition 
of some of the species. The use of various 
mutagenic agents, such as irradiation, led to 
the formation of new forms or strains which 
are often markedly different in their nutrient 
requirements and biochemical activities from 
the mother cultures. 

According to this concept, in the classifi- 
sation of a group of living organisms, no 
single feature can be taken as the predomi- 
nant character. Only when this is combined 
with a group of other characters is one able 
to separate the group into subgroups, no- 
tably genera and species. In selecting a char- 
acter, no matter what its importance in the 
primary subdivision of a group of actino- 
mycetes, one may begin with color; or 
structure of aerial mycelium; or certain bio- 
chemical reactions, which may comprise 
proteolytic activities, utilization of carbo- 
hydrates, production of antibiotics, or phage 
sensitivity. The important thing is to select 
a group of properties to characterize each 
species, with fewer characters, perhaps only 
one, such as antibiotic production, charac- 
terizing varieties. One always encounters, of 
course, the intermediate forms between the 
species. Hach investigator will have to decide 
upon the basis of the combination of charac- 
ters whether to place an unknown culture 


with one species or another. Thus the concept 
of species-group or section has come into 
being. As a further illustration one may take 
S. griseus and S. griseinus, two species be- 
longing to the S. griseus group; both are non- 
chromogenic; the color of the aerial myce- 
hum of both is similar; they are both similar 
morphologically; yet they are different from 
the standpoint of carbon utilization, phage 
sensitivity, and antibiotic production. 

Flaig and Kutzner (1954), Kutzner (1956), 
Baldacei (1959), and numerous others em- 
phasized both physiological and morphologi- 
cal criteria. Gause et al. (1957) emphasized 
the color of substrate and of aerial mycelium 
as well as morphology of sporulating hyphae. 
Numerous new species and varieties were 
described, although very few prior named 
species were discussed or placed into their 
system of classification. Many of these spe- 
cies and varieties are no doubt synonymous 
with previously described forms. 

With streptomycetes, the species are 
linked together so gradually that it is very 
difficult to say where one species ends and 
another begins. The creation of ‘‘sections,”’ 
“oroups,” or ‘‘series’”’ to occupy an inter- 
mediate place between genera and species 
may help in clarifying relationships, but it 
does not do away entirely with the poten- 
tial confusion in the creation of new species, 
especially when the relation of such species 
to those already established is not sufficiently 
understood. This confusion has led some in- 
vestigators to question ‘‘whether the species 


‘ 


concept is tenable in microbiology, and if it 
is not, what we are to substitute for it.” It 
has even been suggested that the idea of 
static species must be abandoned in favor of 
something more elastic. 

Even now, after many additional data 
have accumulated concerning the morphol- 
ogy of the actinomycetes, and after these 
organisms have been separated into a num- 
ber of genera, there is still no general agree- 
ment concerning characterization of species. 


SPECIES CONCEPT IN RELATION TO ACTINOMYCETES 9 


Krassilnikov (1949) insisted that the shape of 
the spore, as seen in the light microscope, 
should be recognized as the major criterion 
for species differentiation. It is doubtful, 
however, whether Krassilnikov’s 
“Jongisporus” and “‘globisporus” types, with 
their many subtypes, can greatly facilitate 
the solution of the problem of species char- 
acterization. The cultural properties of these 


various 


organisms still offer some of the most im- 
portant criteria for species differentiation. 
There is also now available sufficient addi- 
tional information concerning morphology, 
such as formation and branching of the 
sporophores, formation and nature of spores, 
and especially the spore surface as shown by 
the electron microscope, to make possible 
the use of these criteria not only for supple- 
mentary but often for major characteriza- 
tion of the species. 

Several factors have thus contributed to 
the confusion in establishing and recognizing 
species of actinomycetes: (a) lack of clearly 
defined morphological characters; (b) great 
variability of these organisms; (¢) occurrence 
of numerous transition types; (d) ease of 
formation of mutants; (e) lack of sufficiently 
recognizable type species; (f) lack of empha- 
sis upon species-groups and upon type cul- 
tures; and (g) insufficient recognition of the 
formation of well-defined chemical 
pounds which could be used as additional 


cOom- 


criteria for species characterization. 

The suggestion that closely related spe- 
cies be placed in ‘‘species-groups”’ or ‘‘aggre- 
gate-species”’ has recently been gaining con- 
siderable attention. Such a unit should be 
characterized by various reproducible prop- 
erties under standard conditions of culture. 
Baldacci et al. (1953, 1956) suggested that 
micromorphological criteria, namely, seg- 
mentation and branching of vegetative my- 
celium, presence or absence of spores, and 
arrangement of sporophores, be used for 
generic classification. The genus Streptomy- 
ces was then divided, on the basis of pigmen- 


tation of the vegetative and aerial mycelium, 
into a number of ‘series,’ each of which 
was further subdivided into species. Gause 
et al. (1957) made use of the ‘‘series’’ con- 
cept and created a number of groups based 
on the pigmentation of the aerial mycelium. 

When so many different cultures of ac- 
tinomycetes can be isolated easily from natu- 
ral substrates, it is but natural that various 
intermediate types should be found and that 
established species should tend to overlap 
one another. If one were to isolate only a 
small number of cultures, it would be simple 
to recognize a few well defined species. But 
when hundreds of similar strains are found 
in nature and when many of them show only 
minor variations from one another, varia- 
tions which are not important enough to 
warrant creation of new species but are 
nevertheless variations from the established 
type, the difficulties mount rapidly (Fig. 2). 

When study is based upon a single strain, 
a particular species may be described as hav- 
ing a yellow or yellowish aerial mycelium. 
Another strain may produce, on the same 
medium, an aerial mycelium only a shade 
different in color from the original type; 
this pigment may be designated as sulfur- 
yellow, cream-yellow, saffron-yellow, or even 
brownish, all other physiological and mor-- 
phological properties being similar. Would 
one be justified in calling such a new strain a 
different species? The answer is definitely 
“no.” One culture may produce a strong 
tyrosinase reaction, and another only a weak 
reaction, as indicated by pigmentation with 
potato, gelatin, and other protein media. 
One would be inclined to accept these as 
mere quantitative variations allowable for an 
established species. This must be recognized, 
since it is well known that had the test been 
repeated in another laboratory, where the 
medium might be slightly different in com- 
position, the method of sterilization of the 
medium different, or the age and origin of 
the inoculum different, 


these variations 


LO THE ACTINOMYCETES, Vol. II 


Uy 


Wey 


: 
(7 
es 
te A 
\ | 
i 


Fiaure 2. Schematic representation of tuft and cluster formation by certain Streptomyces species 
(Reproduced from: Shinobu, R. Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sei. 7, 1958). 


might have been sufficient to account for the 
minor differences in the color of mycelium 
or in the pigmentation of the medium. But 
what is one to do when the original culture 
is recorded as producing a yellow aerial my- 
celium on a given medium, whereas the new 
isolate gives a buff or brown mycelium? The 
answer would be that if all the other recog- 
nizable properties are the same or similar, 
this would be nothing more than a variant. 
Were one to plate out a single culture and 
pick a large number of colonies, similar varia- 
tions could no doubt be observed. 
Unfortunately, it has frequently been 
found much easier to assign undue impor- 
tance to these variations and designate a 
freshly isolated culture as a new species. 
Some justification for this attitude has been 
found in the fact that the new culture may 
possess an important economic property, 
such as the production of a new antibiotic. 
It is largely for this reason that within the 
last 15 years more ‘‘new”’ species have been 
created than in all the previous 75 years 


since Ferdinand Cohn first deseribed his 
Streptothrix. 


Requirements for Adequate Species De- 
scriptions 


In accordance with the rules of the Inter- 
national Code of Nomenclature of Bacteria 
and Viruses, certain procedures must be fol- 
lowed in describing bacterial species. These 
are summarized by Ainsworth and Cowan 
(1954) as follows: 


The name must be effectively published. 

The name must be validated by a concise de- 
scription of the diagnostic features of the new 
isolate. 

The etymology of the name should be ex- 
plained. 

No Latin diagnosis is required. 

When descriptions are reported in a language 
unfamiliar to the majority of workers, it is recom- 
mended that the authors simultaneously publish 
the diagnosis in a more familiar language. 

Subcultures of the type strains should be de- 
posited at one or more of the national culture 
collections. 


Unfortunately, these simple rules have 


SPECIES CONCEPT IN RELATION TO ACTINOMYCETES 11 


not always been adhered to. Numerous 
names of actinomycetes are reported in the 
literature with no descriptions whatever. 
Some of the descriptions have been published 
partly in languages not generally accessible, 
or in the form of patents, or even as news 
announcements in trade or popular journals. 

Although every effort has been made in 


this treatise to include all species that have 
been adequately described, numerous forms 
must be listed as “incompletely described” 
(Chapter 13). Various names are listed for 
which not even an inadequate description is 
available, the temptation to name a culture 
as a new organism, in order to claim the 
discovery, being too great. 


Chapter 


) 


~_ 


The Genus Actinomyces 


The genus Actinomyces comprises anaero- 
bie or microaerophilic organisms. They are 
mostly pathogenic in nature. The pathogenic 
forms are nonacid-fast, nonproteolytic, and 
nondiastatic. These have been isolated from 
granules in the pus of morbid tissues of a 
human and animal disease known as actino- 
mycosis. They produce no filterable stages 
and show no serological reactions with other 
genera. 

There are also on record observations con- 
cerning the occurrence in various natural 
of nonpathogenic, mesophilic, 
anaerobic actinomycetes that can with full 
justification be included in this genus. Al- 
though few of these have been sufficiently 
studied, one such species is included. The 
saprophytic forms may be proteolytic, ac- 
tively fermentative, and may possess marked 


substrates 


reducing properties. 

The natural relationship of this genus to 
the other genera of the actinomycetes, based 
primarily upon morphological and cytologi- 
cal studies, has recently been examined by 
Bisset (1959). 


Classification of the Genus Actinomyces 


I. Pathogenic forms or forms isolated from 
pathogenic specimens. 
1. Colonies soft, smooth, uniform, not adher- 
ent to the medium. No aerial hyphae. 
a. Causative agent of certain animal dis- 
eases. 
lL. Actinomices bovis 
b. Isolated from human saliva and carious 
teeth. 
8. Actinomyces odontolyticus 


2. Colonies tougher in texture and warted in 


12 


appearance, adherent to medium. Aeria 
hyphae rare. 

a. Hyphae gram-positive and stain faintly 
with hemotoxylin. Causative agent of 
certain human diseases known as actino- 
mycosis. 

6. Actinomyces tsraelii 
al. Related form. 

4. Actinomyces discofoliatus 
Hyphae in pus granules stain with basic 
stains. Cause of actinomycosis in cats 
and dogs. 

2. Actinomyces baudetii 
Il. Nonpathogenie forms. 

1. Occurs in human mouth. 
7. Actinomyces naeslundit 

a. Related form. 

3. Actinomyces cellulitis 
2. Occurs in ground waters. 
5. Actinomyces hvidhansent 


According to Thompson (1950), there are 
two distinet species of anaerobic organisms 
that should be included in the genus Actino- 
myces: A. bovis which is responsible for most 
vases of lumpy jaw in cattle, and A. ¢sraeliz 
which causes most of the typical infections 
in man. This separation of the genus agrees 
with the concepts of other investigators. 
One strain of A. israeliz was recovered from 
a bovine source, and it was suggested that 
some bovine infections may be due to A. 
tsraeliz. On the other hand, the work of Holm 
(1951) and Lentze (1948) indicates that a 
small number of human infections may be 
due to A. bovis. 

Cummins and Harris (1958) fully sup- 
ported the conclusions of Erikson (1940) 
and Thompson (1950) that bovine and hu- 
man strains of Acténomyces are distinct. On 


THE GENUS ACTINOMYCES 13 


the basis of their chemical data, they sug- 
gested that there was very little justification 
for placing bovine strains even in the same 
genus with the strains of A. zsraeliz. Of the 
12 strains received as A. bovis, two were 
identical with the human strains, two showed 
a cell-wall pattern unlike anything hitherto 
recorded, two appeared to be corynebacteria, 
and the remaining six formed a homogene- 
ous group which seemed to be closely related 
to lactobacilli. If cell-wall composition is to 
be considered as any guide to the classifica- 
tion of these strains, the criteria used for the 
identification of A. bovis are insufficient and 
many of the investigators who identified the 
strain were not properly qualified to do so. 

Thompson and Lovestedt (1951) isolated 
cultures from the mouths of 24 patients. In 
addition to two positive cultures of A. 
israeliz, nine of the cultures comprised an 
organism which grew under both aerobic and 
anaerobic conditions. They considered the 
latter to be a saprophyte found in the mouth, 
frequently confused with A. The 
name A. naeslundii was proposed for these 
cultures. 

Howell et al. (1959) made a comparison of 
200 strains of Actinomyces isolated from the 


rsraelit. 


oral cavity in the absence of actinomycosis, 
and 11 isolated from actinomycotic lesions. 
These strains were of two main types, one 
corresponding to the organisms described 
under the name A. naeslundi7, and the other 
essentially identical to those isolated from 
lesions, which should be designated as A. 
israelit. They recommended that A. naes- 
lundit Thompson and Lovestedt be accepted 
as the proper name for the rapidly growing 
facultative type of Actiénomyces. 

One may finally report the results of a 
comparative study (Pine et al., 1960) of 11 
bovine strains of Actinomyces isolated from 
typical cases of lumpy jaw and 15 human 
strains which had identified as A. 
and A. naeslundi. Of the 
strains, one was a typical A. zsraeliz, whereas 


been 


rsraeliz bovine 


the remaining strains formed a homogeneous 
group of fast catalase-negative 
diphtheroids which invariably failed to form 


erowing, 


a true mycelium im vitro; they were thus 
different from both A. 
The last 
classical A. bovis. They produced two kinds 


israelii and A. 
10 strains comprised the 


naes- 
lundii. 


of colonies, depending on the medium: one 
smooth colony, identical to that of Coryne- 
bacterium acnes, and one rough similar to 
that of A. zsraelia but with no mycelium. 
They were anaerobes, forming acid from glu- 
cose but none from xylose, raffinose, or man- 
nitol; nitrates were not reduced and starch 
was rapidly hydrolyzed. They were less path- 
ogenic for animals than human strains, but 
induced lesions in which actinomycotic my- 
celial clumps were formed. The A. 
strains were also anaerobes; they formed 


rsraelar 


acid from glucose, usually from xylose and 
mannitol, and less often from raffinose; ni- 
trates were sometimes reduced to nitrites, 
and starch was poorly hydrolyzed if at all. 
A. naeslundii strains were facultative anaer- 
obes and formed acid from glucose and 
raffinose, but none from xylose or mannitol; 
nitrates were reduced to nitrites and starch 
was poorly hydrolyzed. Micromanipulative 
methods for the study of microaerophilic 
organisms have been examined by Erikson 
(1954); the catalase reaction of A. bovis was 
reported by Suter (1956). 

According to Emmons,* there is little 
value in presenting as valid all the following 
species until they have been studied carefully 
in pure culture. He suggested to accept only 
A. bovis, A. israelii, A. 


* 


baudetii, and A. 
naeslundi. He went so far as to suggest that 
the staining reactions of A. 
hardly sufficient for its differentiation. 


baudetiz are 


Descriptions of Species of Actinomyces 


1. Actinomyces bovis Harz (Harz, C. O. 
In Bollinger, O. Centr. med. Wiss. 15: 485, 


* Personal communication. 


14 THE ACTINOMYCETES, Vol. II 


Figure 3. A. bovis, branching mycelium; cul- 
tured from human tonsils, X 1000 (Reproduced 
from: Emmons, C. W. Puerto Rico J. Public 
Health Trop. Med. 11: 720, 1936). 


1877; Jahr. Miinch. Thierarzeneisch 5: 125, 
LSA): 

Actinomyces bovis was the first authentic 
actinomycete described as a causative agent 
of disease; it is natural, therefore, that it 
should have a number of synonyms. These 
are given here, without any guarantee that 
the list is complete. 

Synonyms: Discomyces bovis Rivolta, 
1878; Bacterium actinocladothrix Afanasiev, 
1888; Nocardia actinomyces de Toni and Tre- 
visan, 1889; Actinomyces hominis Bostroem, 
1890; Streptothrix actinomyces Rossi-Doria, 
1891; Cladothrix bovis Macé, 1891; Oospora 
bovis Sauvageau and Radais, 1892; Actino- 
myces albidoflavus Rossi-Doria, 1891; Actino- 
myces sulphureus Gasperim, 1894; Nocardia 


bovis R. Blanchard, 1895; Streptothrix israeli 
Ixruse, 1896; Cladothrix actinomyces Macé, 
1897; Streptothrix actinomycotica Foulerton, 
1899; Discomyces bovis R. Blanchard, 1900; 
Streptothrix spitzi Ligniéres, 1903; Sphaeroti- 
lus bovis Engler, 1907; Cohnistreptothrix 
israeli Pinoy, 1911; Actinomyces israeli Vuil- 
lemin, 1931. See also Baldacei (1937). 

Morphology: Grows in the form of sulfur- 
colored granules in the pus of cases of ac- 
tinomycosis. The radiating hyphae are cov- 
ered with extraneous material deposited by 
the host to form clubs. Organism is gram- 
positive, nonmotile, nonacid-fast. Colonies 
are dull white in color, only slightly ad- 
herent to the medium. No aerial hyphae. 
Mycelium undergoes fragmentation very 
rapidly into V- and Y-forms. Extensive 
branching is rare. Hyphae less than 1 » in 
diameter (Fig. 3). 

Semisolid media: Growth excellent, espe- 
cially with paraffin seal. No soluble pigment 
produced. 

Gelatin: Growth secant, flaky. No lique- 
faction. 

Liquid media: Occasional turbidity with a 
light, floceulent growth. 

Egg or serum media: No proteolytic ac- 
tion. 

Milk: Turns acid; no coagulation and no 
peptonization. Sometimes there is no growth. 

Sugar utilization: Acid from glucose, su- 
crose, and maltose; no acid from salicin or 
mannitol. 

Temperature: Optimum 37°C. Does not 
grow at 22°C. Killed at 60°C. 

Oxygen requirement: Anaerobic to micro- 
aerophilic. Grows readily in an atmosphere 
of CO,.. Bovine strains are more oxygen- 
tolerant on egg or serum media than strains 
of human origin. 

Viability: Pure cultures do not live more 
than 10 to 14 days. On Dorset’s egg medium, 
they may survive in an ice chest for 3 to 4 
weeks. 

Habitat: Originally found in lumpy jaw of 


THE GENUS ACTINOMYCES 15 


cattle. Usually found in and about mouths of 
animals. 

Remarks: King and Meyer (1957) re- 
cently suggested that in order to implement 
proper identification of A. bovis, certain se- 
lected differential criteria, such as catalase 
test, litmus milk reactions, and the utiliza- 
tion of xylose, salicin, and raffinose, can be 
used. Slack and Moore (1960) suggested the 
use of fluorescent antibody formation for the 
further identification of this organism. 


2. Actinomyces baudeti Brion, 1942 (Brion, 
G. de. Rev. de Méd. Vétér. 91: 157, 1942; 
Brion, G. de, Goret, and Joubert. Proc. VI 
Congr. Intern. Patol. Comp., Madrid 1: 48, 
1952). 

Morphology: Granules from histological 
preparations show tangled, radiating hy- 
phae; ends of hyphae rounded and ovoid, 
forming a crown. Hyphae take basic stains. 
Mycelium composed of slender hyphae, 0.2 
to 0.4 pw. Nonseptate. Ends swollen and 
rounded. Copious branching. In artificial 
media hyphae are frequently short, rarely 
exceeding 20 uw in length. 

Agar colonies: Dull, whitish granules ad- 
hering slightly to the medium. 

Liquid media: A sediment of white gran- 
ules is produced. 

Gelatin: No liquefaction. 

Blood serum: In 4 to 5 days, surface cov- 
ered with white granules which are the size 
of a pin head. 

Serum media: No proteolytic action. 

Brain extract: Growth favored in some 
media. 

Indol: Production slight. 

Sugar utilization: Acid from glucose, su- 
crose, and starch. 

Oxygen demand: Anaerobic to microaero- 
philic. 

Optimum temperature: 37°C. 

Pathogenicity: Pathogenic when inocu- 
lated into dogs, rabbits, and guinea pigs 
(forms subcutaneous abscesses). 


Source: Isolated from various types of le- 
sions in cats and dogs. 


3. Actinomyces cellulitis (Linhard, 1949) 
nov. comb. (Linhard, J. Ann. inst. Pasteur 
76: 478, 1949). 

Synonym: Actinobacteriwm cellulitis Lin- 
hard. 

Morphology: Polymorphic rods, showing 
primary, secondary, and sometimes tertiary 
branching. Length 5 to 7 uw, diameter 0.6 wu. 
Nonmotile. Gram-positive. 

Agar media: Colonies lenticular. No gas. 

Glucose broth No. turbidity. 
Abundant growth, settling to bottom. 

Gelatin: No liquefaction. 

Milk: Unchanged. 

Serum: Serophilic, but can be adapted to 
serum-free media. 

Nitrate reduction: Positive. 

Oxygen demand: Anaerobic and micro- 


cultures: 


aerophilic. Colonies produced at 4 to 5-mm 
depth in agar media. 

Reduction: Does not reduce neutral red or 
safranin. 

Carbon utilization: Positive utilization of 
glucose, fructose, maltose, galactose, and 
sucrose. Produces volatile acids (propionic 
and formic). Production of gas may suggest 
either a contaminated culture or the absence 
of an Actinomyces. 

Pathogenicity: Nonpathogenic. 

Habitat: Oral cavity of man. 


4. Actinomyces discofoliatus — (Griiter, 
1932) Negroni (Negroni, P. Mycopathol. 1: 
81-87, 1938-1939). 

Morphology: Deep colonies in semisolid 
glucose agar are whitish, lens-shaped, crossed 
or forming dihedral angles; margins of colo- 
nies regular; consistency of colonies slimy. 
Bacteria-like entities measuring 3 to 4 u to 
10 to 15 w by O.8 yw, occurring as isolated 
elements or V- or Y-shaped elements. Com- 
pact colonies in hanging-drop cultures. The 
filaments have a tendency to dichotomous 


16 THE ACTINOMYCETES, Vol. II 


FicurReE 4. A. 
Institute of Pathology). 


branching, with prevailing development of 
one branch. 

Glucose agar: Discoid, moist, and brilliant 
colomes; shghtly elevated in the central part 
with nearly regular margins. 

Gelatin: No liquefaction. 

Glucose broth: Slimy sediment and some- 
times a shght turbidity. The medium be- 
comes clear at the end of 8 to 10 days. 

Carbon sources: Acid but no gas from glu- 
cose, maltose, fructose, lactose, sucrose, and 
inulin; very little or no acid from mannitol. 

Starch: Not attacked. 

Sucrose: Not inverted. 

Nitrate reduction: Negative. 

H.S: Formed. 

Indol: Shght quantity produced. 

Fats: Shghtly attacked. 

Olive oil: Not attacked. 

Optimum temperature: 37°C. 

Oxygen demand: Facultatively anaerobic. 

Remarks: Vitality weak. Deep cultures in 
semisolid media die if held for longer than 8 


le dey wal 


to 10 days at 37°C, or for longer than 30 


minutes at 60°C. Exposure for longer than 


israelii, grown anaerobically in veal infusion agar, X 975 (Courtesy of Armed Forces 


a few minutes in dilute mineral acids kills 
the organism. The organism can be kept 
alive for 2 to 3 months if cultures are kept 
in an ice chest, in a dried state, or under 
vacuum. 

Habitat: Lachrymal concretions and hu- 


man actinomycotic lesions. 


5. Actinomyces hvidhanseni (Hvid-Han- 
sen, 1951) nov. comb. (Hvid-Hansen, N. 
Acta Pathol. Microbiol. Scand. 29: 335-338, 
1951). 

Synonym: Actinomyces israeli Hyid-Han- 
sen. 

Morphology: Gram-positive, nonacid-fast, 
nonmotile. Polymorphic, bent, and often 
branched rods. Obligately anaerobic. 

Meat liver agar: Colonies circular or ir- 
regular, often in the form of bodies bounded 
by four concave surfaces meeting in four 
acute vertices, of highly varying size and of 
a pale pink color. Surface colonies circular, 
convex, grayish-white or white; transparent 
S-colonies of a butyrous, viscous, but not 
mucous consistency. 

Meat liver broth: Diffuse growth at first, 


THE GENUS ACTINOMYCES 17 


followed rapidly by a fairly voluminous pale 
pink, homogenous precipitate. 

Thioglycollate medium: Growth either 
diffuse, netlike, or dispersed and granular. 

Gelatin: Liquefied. 

Milk: Coagulated in 24 to 48 hours and 
peptonized in 3 weeks. 

Blood: All strains hemolyze human blood 
on solid media but do not form a soluble 
hemolysin. 

Sugar utilization: Galactose, fructose, and 
glycerol vigorous; inulin, maltose, mannitol, 
starch, duleitol, and 
somewhat less readily; xylose and arabinose 
not at all. 

Reduction: Some strains form a little hy- 
drogen sulfide. Sulfites and sulfates are not 
reduced. Nitrates reduced to nitrites and in 


saccharose, lactose 


some cases to ammonia. Safranin, phenosaf- 
ranin, or neutral red reduced. Most 
strains decolorize methylene blue in 4 to 24 


not 


hours; some do not. 

Temperature: Optimum 37°C. Heating to 
50-60°C for 15 minutes injurious. 

Remarks: All strains catalase-positive. All 
produce ethyl alcohol, aldehyde, acetone, 
ammonia. A faint indol reaction is found in 
alkaline distillate. The presence of volatile 
acids, tartaric acid, and lactic acid has been 
demonstrated, but not succinic acid. Pro- 
plonic acid and formic acid in ratios of from 
3 to 1 up to 20 to | for the six strains ex- 
amined. 

Habitat: Ground water. 

Remarks: Kalakoutski (1960) found an- 
aerobic actinomycetes in natural waters and 
in the air of apartments occupied by man, 
but not in the soil. 


6. Actinomyces israelii (Kruse) Lachner- 
Sandoval, 1898 (Wolff, M. and Israel, J. 
Arch. pathol. Anat. 126: 11, 1891). 

Synonyms: Streptothrix 
1896; 
Actinomyces bovis Wright, 
Prumpt, 1906; 
israeli Sampietro, 1908; 


Kruse, 
Gedoelst, 1902; 
1905; Discomy- 


rsraeli 
Discomyces israelr 
ces bovis Actinobacterium 


Cohnistreptothria 


israeli Pinoy, 1913; Nocardia israeli Castel- 
lani and Chalmers, 1913; Brevistreptothrix 
israeli Ligniéres, 1924; Proactinomyces israeli 
Jensen, 1931; Corynebacterium israeli Lentze, 
1938; Actinomyces israeli var. indo-sinensis 
Reynes, 1947. 

Morphology: Large, club-shaped forms 
are seen in morbid tissues. Substrate myce- 
lium consists of rapidly septating and spor- 
ulating hyphae. The branches may extend 
into the medium in long filaments or may 
exhibit fragmentation and characteristic an- 
gular branching. Hyphae occasionally sep- 
tate, but no definite spores are formed. 
Colonies exhibit a considerable degree ot 
polymorphism, but no stable variants have 
been established. Colonies are tougher in 
texture than those of A. bov7s. Old colonies 
warted in appearance (lig. 4). 

Gelatin: Growth scant, flaky. No liquefae- 
tion. 

Liquid media: Growth in form of white 
compact colonies or granular sediment. Me- 
dium shows no turbidity, usually remaining 
clear. No gas and no odor. 

Pigments: No soluble or insoluble pig- 
ments. 

Ege media: No proteolytic action. 


Milk: but 


not clot. No peptonization. Frequently no 


Becomes acid, usually does 
growth. 

Starch: Shght hydrolysis. 

Oxygen requirement: Anaerobic. 

Nitrate reduction: Generally negative. 

Sugar utilization: Greater ability to utilize 
sugars than A. bovis. Acid but no gas from 
glucose, galactose, lactose, fructose, maltose, 
raffinose, sucrose; no acid from inulin. 

Hemolysis: Slight to marked. 

Serological reactions: Lack of serological 
affinity with A. bovis. 

Temperature: Optimum 37°C. Destroyed 
at 55-60°C in 30 minutes. 

Habitat: Dental caries, tonsils, and natu- 
ral cavities of man and animals. Chief etio- 
logical agent of human actinomycosis, de- 


Re) THE ACTINOMYCETES, 


J 
oe’ 


Figure 5. A. israelii (Reproduced from: Pré- 


Vota Au RR tas Intern. Congr. Microbiol., Symp. 
Actinomycetales, Rome, 1953, p. 45). 


scribed first by Wolff and Israel (1891) and 
later by Wright (1905). 

temarks: Vitality weak. Cultures no 
longer viable after 8 to 10 days. Erikson and 
Porteous (1953) succeeded in obtaining good 
growth by continued subculture in a medium 
containing 99 parts of 1 per cent casein hy- 
drolyzate and 1 part of heart broth and 0.5 
per cent glucose. Antigenic structure of or- 
ganism has been recently studied by Itwa- 
pinski (1960). 

According to Grootten (1934) 
ism is highly polymorphic. Rods varying in 
length are formed in young culture. They are 
straight or slightly curved, with round 
oval extremities. Occasionally, long or even 
filamentous forms are found. Some of the 
filaments end in small spherical or pear- 
shaped swellings. It does not form spores. 
t does not grow in the upper 
5- to 10-mm zone; below that zone, it forms 
a layer of 2 to 4 mm with numerous small 
in the deeper layers, the colonies 
attain diameters of 2 to 3 


, the organ- 


In agar tubes, 1 


colonies; 

are fewer, 
No 
Liquid media remain clear. 
nonproteolytic; milk is not coagulated. Blood 


but may 
mm. gas and no odor are produced. 


The organism is 


Vol. II 


is rapidly hemolyzed. It does not grow on 
potato plugs, except poorly when glycerin- 
ated. It slowly attacks glucose, lactose, mal- 
tose, sucrose, and mannitol. It does not grow 
in glucose-gelatin medium. Animal infection 
is obtained by introducing a culture into the 
peritoneum of rabbits. 

Negroni (1954) deseribed <A. 
further detail. 


israelit in 
Deep colonies in semisolid 
glucose agar are globous, 1 to 2 mm in 
diameter, whitish, opaque, and with an ir- 
regular surface. Colonies are of a 
consistency and cannot be homogeneously 
suspended in water. On glucose or glycerol 
agar slants, the colonies are elevated, mam- 


cheesy 


milated, and whitish, with moist and_ bril- 
liant surface and irregular margins. Sub- 
merged mycelium is well developed. The 


colonies have a cheesy consistency and can 
sasily be removed from the medium with a 
platinum loop (Fig. 5). 

According to Erikson and Porteous (1955), 
the conversion of a “rough” typical strain of 
A. israelii to a ‘“‘smooth” soft form more 
tolerant of oxygen is a result of the physical 
trapping within the mycelium of a few alien 
facultative anaerobes, usually staphylococci. 


Actinomyces naeslundit Thompson and 
Lovestedt, 1951 (Thompson, L. and Love- 
stedt, S. A. Proc. Staff Meet. Mayo Clinic 
26: 169, 1951). 

Morphology: Organism forms small, whit- 
ish, firm colonies. Mycelial branching, but no 
segmentation. Not acid-fast. 

Artificial media: Good growth. 

Hormone agar: Rough and smooth colo- 
nies, | to 2 mm in diameter, after 4 days. 
Surface of colonies varies from smooth to 
nodular to wrinkled. Consistency varies from 


butyraceous to tough and adherent. Colo- 
nies are opaque, with color varying from 
white to cream. 


Glucose brain broth: Growth rapid and 


abundant. Acid produced. 
Gelatin: Growth slow. No liquefaction. 
Starch: Not hydrolyzed. 


THE GENUS ACTINOMYCES 19 


Milk: Growth scant or absent. 

Aerobiosis: Grows both under aerobic and 
anaerobic conditions, somewhat better aero- 
bically. 

Temperature: Optimum at 37°C; 
growth at 32°C. 

Pathogenicity: Nonpathogenic. 

Habitat: Human mouth. Considered to be 
a saprophyte found in the mouth and fre- 
quently confused with A. zsraeliz. 


some 


8. Actinomyces odontolyticus Batty, 1958 
(Batty, I. J. Pathol. Bacteriol. 75: 455-459, 
1958). 

Morphology: At first, the organism ap- 
pears in the form of short rods subdivided 
by one or two transverse septa. Later, these 
rods gradually elongate until a septate sub- 
mycelium is produced. At the end of these 
filaments, globular ‘“‘initial cells’? are pro- 
duced which germinate to produce a non- 
septate secondary submycelium, which soon 
commences to break up. Finally, in a week 
to 10 days, small spores commence to form 
singly upon short side branches. The size of 
the mature spores varies greatly in different 
strains. 

Appearance of colonies: Colonies are usu- 
ally few in number with an initial appearance 
similar to those of a-hemolytic streptococci 
of comparable age. Later, they develop a 
dark red hemin-like pigment, 
tinguishable. At and after this stage the 
colonies are exceedingly difficult to subcul- 
ture. After prolonged artificial culture the 
organisms can be subcultured at any stage. 
Attempts to isolate the organism upon horse 
serum agar or nutrient agar are usually un- 
successful, but after several subcultures a 


sasily dis- 


profuse growth of small convex nonpig- 
mented colonies is obtained on both these 
media. All strains grow equally well under 
aerobic and conditions at all 
stages in their life cycle; in agar stab cultures 
a filiform growth is obtained throughout the 
line of inoculum. Growth in peptone broth is 
sparse, but in this medium enriched with 


anaerobic 


odontolyticus, various stages of 


Ficure 6. A. 
culture development (Reproduced from: Batty, I. 
J. Pathol. Bacteriol. 75: 455-459, 1958). 


yeast extract, a characteristic glutinous ropy 
sediment is produced which disperses to 
give an even turbidity (Fig. 6). 

No strain produces catalase, oxidase, in- 
dole, hydrogen sulfide or acetylmethylear- 
binol; all are methyl red-negative and all fail 
to ferment fructose, trehalose, 
starch, inulin, dextrin, 


rhamnose, glycerol, dulcitol, and salicin. A 


maltose, 

glycogen, xylose, 
few strains ferment sucrose, galactose, arabi- 
nose, or mannitol, with the production of 
acid but no gas. About half the strains pro- 
duce ammonia from peptone, acidify and 
coagulate litmus milk, and are tolerant of a 
concentration of 1 in 4000 potassium tellu- 
rite. Some strains hydrolyze urea. None 
liquefy gelatin, Loeffler’s medium, or coagu- 
nitrate to 


lated egg medium. All reduce 


nitrite within 18 hours. 


20 THE ACTINOMYCETHES, Vol. II 


Habitat: Human saliva in deep dental 
caries. 

Remarks: This organism 
bovis in its life cyele. 


resembles A. 


Incompletely Described Forms of Ac- 
tinomyces 


In addition to the above well described 
and readily recognizable forms belonging to 
the genus Actinomyces, numerous other an- 
aerobic organisms have been listed in the 
literature. Some of these organisms are no 
doubt strains of the well described forms and 
their names would be in synonymy. Others 
may represent distinct species. 

It is of particular interest to cite, in this 
connection, the ideas of Prévot (1957), who 


considered Actinomyces bovis as an aerobic 
organism and, therefore, the genus Actino- 
myces as an aerobic group. He suggested 
that Actinomyces israelii represent the an- 
aerobic group, and the generic name Actino- 
bacterium Haas, 1906 (Syn. Cohnistrepto- 
thrix Pinoy, 1913) be given priority for desig- 
nating the anaerobic forms. Prévot (1957) 
divided the genus Actinobactertum into six 
species: (1) A. zsraeliz, (2) A. meyers, (3) A. 
abscessus, (4) A. liquefaciens, (5) A. cellu- 
litis, and (6) A. propionict. 

In addition to the above, numerous other 
anaerobic forms have been described, such 
as A. canis Levy, 1899; A. interproximalis 
Fennel, 1918; and others. Some additional 
names will be found in Chapter 13. 


=) 


Chapter 3 


The Genus Nocardia 


Characterization of Genus 


The genus Nocardia represents a 
of aerobic actinomycetes which includes both 
pathogens and saprophytes. The relation- 
ship of this genus to, and possible overlap- 
ping with, the genus Mycobacterium, on the 
one hand, and the genus Streptomyces, on the 
other, have already been discussed (Volume 
I). Numerous cultures of nocardiae have 
been isolated from human and animal infec- 
tions, and claimed to be the causative agents 
of the particular disease. The fact, however, 
that a culture of an organism has been iso- 
lated from a lesion of a man or an animal is 
no proof that it is primarily responsible for 
the particular disease; it may actually be a 
secondary invader or a member of a mixed 
infection. Some species of Nocardia are def- 


group 


initely associated, however, with certain 
diseases, or have at least been isolated from 
infected tissues. This gave origin to the term 
“nocardiosis,’ descriptive of these disease 
conditions. 

The colonies produced by nocardiae are 
either smooth, or rough and much folded; 
they are either of a soft or dough-like con- 
sistency, or compact and leathery, especially 
in early stages of growth. Many species of 
Nocardia do not form any aerial mycelium; 
some give rise to a limited aerial mycelium 
which may structurally be similar to that of 
the substrate mycelium; still others may 
produce aerial hyphae and spores which may 
be indistinguishable from those of Strepto- 
myces and are thus responsible for various 


cases of overlapping between these two 
genera. 
Nocardias multiply by concentration 


and segmentation of the protoplasm within 
a filamentous cell, followed by dissolution of 
the cell membrane. The fragmented portions 
of the mycelium usually develop into fresh 
mycelium under favorable conditions, either 
by germ tubes or by lateral budding. Strep- 
tomycetes produce true spores or conidia, the 
vegetative mycelium not segmenting spon- 
taneously into bacillary or coecoid forms, but 
remaining nonseptate and coherent even in 
old cultures, thus producing the characteris- 
tic tough textured, leathery growth. In no- 
cardiae, the aerial mycelium represents an 
extension upward of the vegetative myce- 
lium; it does not exhibit any differentiated 
protoplasm and is sterile and abortive. When 
a streptomycete has lost the capacity of 
producing aerial mycelium, a form analogous 
to that of a nocardia may result, except for 
the structure the mycelium and the 
capacity of the degenerated streptomycete 
to regain the lost capacity. It is occasionally, 
therefore, a matter of personal preference 
whether to place a freshly isolated culture in 


of 


one genus or another. Some nocardiae are 
acid-fast or partially acid-fast, and others 
are not. 

The mode of branching of the substrate 
mycelium (see Volume I, Chapter 5), the 
biochemical properties (proteolytic and sero- 
logical activities), and chemical nature of the 
cell walls of nocardiae appear to distinguish 
them from the streptomycetes. Hoare and 


22 THE ACTINOMYCETES, Vol. II 


Work (1957) have shown that these genera 
‘an be differentiated by the configuration of 
the diaminopimelic acid present in whole cell 
hydrolysates; streptomycete cell walls con- 
tain the L-isomer, whereas nocardiae cell 
walls contain the pL-isomer. Cummins and 
Harris (1958) reported that the presence or 
absence of arabinose in the hydrolysates of 
the intact organisms can also be used to 
identify them; nocardiae cell walls contain 
arabinose, whereas streptomycete cell walls 
do not. The sensitivity of most Streptomyces 
species, but not of nocardiae, to the action of 
lysozyme on their cell wall preparations, 
studied by Sohler, Romano, and Nickerson 
(Volume I, p. 159), provides further criteria 
for distinguishing between members of these 
two genera. Studies of infrared absorption as 
a taxonomic criterion (Riddle et al., 1956) 
has also been suggested.* 

In view of the overlapping between certain 
forms placed for convenience in either one 
genus or the other, the separation of atypical 
strains of Nocardia or Streptomyces by mor- 
phology or fermentation tests alone may be 
difficult, as pointed out by Gordon and 
Mihm (1957). 

The genus Nocardia has been described in 


the last edition of Bergey’s Manual as 
follows: 
“Slender filaments or rods, frequently 


swollen and occasionally branched, forming 
a mycelium which, after reaching a certain 
size, assumes the appearance of bacterium- 
like growths. Shorter rods and coccoid forms 
are found in older cultures. Conidia not 
formed. Stain readily, occasionally showing ¢ 


slight degree of acid-fastness. Nonmotile.t 


* Personal communication from Dr. N. M. 
McClung. 

7+ The existence of motility among the nocardias 
was considered by Jensen (1953) as indisputable, 
and this really is not surprising in view of the 
numerous observations on motility in the closely 
related coryneform bacteria. The species in the 
order Actinomycetales cannot any longer be re- 


garded as constantly nonmotile (Fig. 14). 


No endospores. Aerobic. Gram-positive. The 
colonies are similar in gross appearance to 
those of the genus J/ycobacterium. Paraffin, 
phenol and m-cresol are frequently utilized 
as a source of energy. 

“In their early stages of growth on culture 
media (liquid or solid), the structure of 
nocardias is similar to that of actinomycetes 
in that they form a typical mycelium; hy- 
phae branch abundantly, the branching 
being true. The diameters of the hyphae vary 
between 0.5 and 1 yu, usually 0.7 to 0.8 pn, 
according to the species. The mycelium is 
not septate. However, the further develop- 
ment of nocardias differs sharply from that 
of actinomycetes; the filaments soon form 
transverse walls and the whole mycelium 
breaks up into regularly cylindrical short 
cells, then into coecoid cells. On fresh culture 
media, the coccoid cells germinate into my- 
celia. The whole cycle in the development of 
noecardias continues for 2 to 7 days. Most 
frequently the coccoid cells are formed on 
the third to fifth day, but in certain species 
they can be found on the second day. 

“The multiplication of nocardias proceeds 
by fission and budding; occasionally they 
form special spores. Budding occurs often. 
The buds are formed on the lateral surface 
of the cells; when they have reached a cer- 
tain size, they fall off and develop into rod- 
shaped cells or filaments. The spores are 
formed by the breaking up of the cell plasm 
into separate portions usually forming 3 to 
5 spores; every portion becomes rounded, 
covered with a membrane and is transformed 
into a spore; the membrane of the mother 
cell dissolves and disappears. The spores 
germinate in the same way as those of ac- 
tinomyces. They form germ tubes which 
develop into a mycelium (Fig. 7). 

“The colonies of nocardias often have a 
paste-like or mealy consistency and can 
sasily be taken up with a platinum loop; they 
spread on glass and occasionally render the 
broth turbid. The surface colonies are 


THE GENUS 


. Fie 
| 
» 
p : 
F as = 4 ‘ 
‘ 4 
ad, ’ 
4 te. ~~ Lf ’ 
, 7 ‘ Ps « 4 
x \ ‘\ at ea A SG eS 
> / 7 Reena 
ay ‘ SF 2 a7 
{ ~ 
N ee Ne 
aN : oe 
ged — ey 
“NN 
te 
Pad 
¥ é 
* 
* 
i 4 g. 
be + % 
4 j 
? én 
ee e * 
— . 
* ia & Cc 
* 
4 : ? 
# 


Ficure 7. N. 


NOCARDIA 23 


opaca: (a) grown for 4 days on n-dodecane and mineral salts; gram stain, X 960; (b) 


same grown 3 days; X 3700; (c) same as (a) but using fat stain, X 1920; (d) two-day growth, X 12,500 


(Reproduced from: Webley, D. M. J. Gen. Microbiol. 11: 425, 1954). 


smooth, folded or wrinkled. Typical nocar- 
dias never form an aerial mycelium, but 
there are cultures whose colonies are covered 
with a thin coating of short aerial hyphae 
which break up into cylindrical oidiospores. * 
Many species of nocardias form pigments; 
their colonies are of a blue, violet, red, 
yellow or green color; more often the cultures 
are colorless. The color of the culture serves 
as a stable character. The type species is 


Nocardia farcinica Trevisan.” 


Classification of Nocardia Species 

De Toni and Trevisan (1889) described 
five species of Nocardia: N. farcinica, N. 
actinomyces, N. foerstert, N. arborescens, and 


N. ferruginea. 


* See the work of Gordon and Mihm (1958). 


Jensen (1932a) found that a number of 
organisms previously described as species of 
Mycobacterium actually belong, on account 
of definite mycelial growth in the initial 
stages of their life cycles, to the genus Nocar- 
dia.* Mycobacterium agreste Gray and Thorn- 
ton and B. mycoides corallinus Hefferan were 
found to be similar to one another and were 
The 


same was true of JJ. salmonicolor den Dooren 


regarded as one species, N. corallina. 


de Jong, which was designated as N.. sal- 


monicolor. Mycobacterium  opacum den 


Dooren de Jong and JM. crystallophagum 
Gray and Thornton proved to be identical 
and were named N. opaca. Mycobacterium 
erythropolis, a closely related form, was des- 


* Generic name Proactinomyces used. 


Ficure 8. N. paraffinae, showing a section of a 
colony covered with mature aerial mycelium (Re- 
produced from: Hirsch, P. and Engel, H. Ber. 
Deut. Botan. Ges. 69: 454, 1956). 


Fiacure 9. Nocardia, strain 70, showing the de- 
velopment of the aerial mycelium on mineral agar 
(Reproduced from: Hirsch, P. and Engel, H. Ber. 
Deut. Botan. Ges. 69: 454, 1956). 


LI; 


THE ACTINOMYCETES, Vol. II 


A. Unstable mycelium (a-form), with 
short mycelium (if formed at all), 
bacterial (diffuse) growth in liquid 
media, bacteria-like colony. 

Stable mycelium (8-form), producing 
long hyphae, colony growth in liquid 
media, and Streptomyces-like type of 
colony. 

Umbreit (1939) modified the system of 


B. 


Jensen as follows: 
I. Partially acid-fast, nonproteolytic, non- 


diastatic; constantly utilize paraffin. 

1. a-Mycelium type: N. opaca,* N. eryth- 
ropolts. 

. B-Mycelium type: 


a. Red-colored: N. polychromogena 
N. asteroides. 

b. Yellow-colored: NV. paraffinae. 
Nonacid-fast forms, constantly diastatic 
1. a-Mycelium type: 

a. Nonproteolytic: N. mesenterica. 

b. Proteolytic: N. actinomorpha. 

2. B-Mycelium type: 
a. Yellow-colored: N. flavescens. 
b. Red to orange: N. maculata. 
Krassiinikov (1938) divided the genus 


Nocardia* into two groups: 
re) 


I. Well developed aerial mycelium, with 


ignated as N. erythropolis. Microbacterium 
mesentericum Orla-Jensen was renamed N. 
mesentericus. 

Jensen divided the genus Nocardia into 
two distinct groups: 

I. Nonproteolytic organisms with strongly 
refractive cells showing a partial acid- 
fastness in milk and sometimes in other 
media; capable of decomposing paraffin. 
Some species of this group form a transi- 
tion to the genus A/ycobacterium. 

II. Mostly proteolytic forms with weakly 

refractive, nonacid-fast cells. This group 
forms a close transition to the forms now 
included in the genus Streptomyces. 

A further separation of the genus was 
based upon the structure of the aerial myce- 
lium. 


Ale 


substrate mycelium seldom producing 
cross walls. The hyphae break up into 
long, thread-like rods. Branches of the 
aerial mycelium produce segmentation 
spores and oidiospores; the latter are eyl- 
indrical with sharp ends. No spirals or 
fruiting branches. This is the same as 
group 8 of Jensen. 

Typical nocardial forms. Mycelium de- 
velops only at early stages of growth, 
then breaks up into rod-shaped and 
coccoid bodies. Smooth and rough col- 
onies, dough-like consistency, similar to 
bacterial colonies. Aerial mycelium not 


formed or only around colonies. 


* Generic name Proactinomyces used. 


THE GENUS NOCARDIA 25 
TABLE | 
Summary of growth characteristics of 18 strains of Nocardia (McClung, 1949) 
Fragmentation 
Organism Scion |branchiae | branching 
Age Type 1 Type 2 Type 3 
hr hy hr hr q % % 

Group I 

WieBie ae SRL We le Sach toh 2 8 16 0 13 70 30 0 

INESOORLOSUUS Hera ee Stef 6 12 0 12 60 30 10 

SU bo ial ene ea 9 15 0 13 50 50 0 

BB eeeaer ete tees er 10 36 0) 14 50 50 0 

N. erythropolis...... ; 7 1] 14 0 14 69 40) 0 

N. polychromogenes... ‘rE 1] 13 0 14 40 30 30 
Group II 

NSO Ree eae ec tes Sate 1] 12 5 19 6 28 66 

‘oleae SA ae meee 10 12 7 1S 5 40 50 

INGETALD Chetan esc 10 14 1 20 5) 30 65 

N. polychromogenes............ 14 30 20 120 Unknown 

IN@rastenovdes:s.<..)... 10 28 96 96 Unknown 
Group III 

ees Met ewer Paes ee 529s ah Sache ee oe: 10 16 4 0 0 0 

Pedal errant erty t ora cat's loc). -yerantontrs 9 1] 6 0 0 0 

tse O08 Sie) fue ae eae a 10 12 S 0) 0 0 

NOE Zeta eaten! Staves ne Seetehehs aust 11 15 6 0 0 0 

WINS) Shh alk o Aleantsi eo eeee ecere ee 10 15 5) 0 0 0 

Hato goa ra Riek Oke i eres ee pate DR a eee 10 13 9 8) 0) 0 

2) = OST O IE T TN Eco cous ys chen epes sis anes Bes Ss 13 7 0 0) 0 


MeClung (1949) divided the genus Nocar- 
dia into three groups: 

I. Scant mycelial development, sparse 
branching. Colonial texture soft, pasty, 
and sometimes mucoid; pigment intra- 
cellular and insoluble. 

Extensive mycelial development, 
straight branches which do not over- 
lap. Colonial texture soft and pasty; 
pigment intracellular and insoluble. 
oxtensive mycelial development, 
fragmentation of hyphae, contorted and 
profusely produced branches which 
overlap. Colonial texture 
‘artilaginous; generally both intracel- 
lular and soluble pigments are pro- 
duced (Figs. 8, 9). 

The pattern in Nocardia fragmentation 
can be separated into three types. In Type 
1, an acute angle is formed in a hypha pre- 
ceding division, which occurs at the apex of 


II. 


III. 


no 


Waxy or 


the bend; following division the new hyphal 
tips grow out parallel to each other. In Type 
2, division occurs in a straight or slightly 
curved portion of a hypha; following di- 
vision, the newly formed ends bend slightly 
and grow past each other. In Type 3, di- 
vision occurs in the parent hypha close to or 
at the juncture of a branch; a new hypha 
may grow from the place of division at 
the base of the branch; the newly formed 
hyphal tip bends and continues to grow. 
Type 1 fragmentation is characteristic of 
Group I, and Type 3 of Group II. Type 2 
occurs in both groups, and Group III lacks 
fragmentation (Fig. 10). 

A summary of the growth characteristics 
of various strains of Nocardia belonging to 
these three groups is presented in Table 1. 

In an attempt to find a group of depend- 
able properties for the separation of the 
genera Nocardia, Streptomyces, and Mvyco- 


26 THE ACTINOMYCETES, Vol. II 


Fieure 10. N. rubra: (above) 24 hr, glycerol 
nutrient agar, methylene blue; (below) same, 
stained with Sudan black B, X 1600 (Reproduced 
from: McClung, N. M. Lloydia 12: 165, 1949). 


bacterium, Gordon and Smith (1955) exam- 
ined 152 cultures labelled Streptomyces and 
99 cultures labelled Nocardia; those strains 
of the latter that formed soft, fragmenting, 
vegetative mycelium were excluded. Of the 
cultures designated as Streptomyces, 83 per 
cent produced an aerial mycelium typical of 
this genus; 13 per cent failed to produce 
aerial mycelium, although a few formed rudi- 
mentary aerial hyphae; inability to form 
spores was considered as a lost property, 
the physiological reactions of the 
strains were the same as in the previous 


since 


group of sporulating cultures; only five 
strains, or + per cent of the cultures possessed 
nocardial properties. Of the cultures desig- 
nated as Nocardia, 68 produced aerial hy- 
phae, varying from rudimentary to luxuri- 
ant, some even forming chains of spores. 
According to their physiological properties, 
24 of these cultures should have been desig- 
nated as Streptomyces. A few of the strains 


could be considered as intermediate between 
the two genera. 

Gordon and Mihm (1957) further reported 
the results of an examination of 219 cultures 
labelled Streptomyces, 214 Nocardia, and 243 


TABLE 2 
Certain physiological and biochemical characteris- 
tics of various strains of two species of Nocardia 


(Gordon and Mihm, 1959) 


N. asteroides N. brasiliensis 
(98 strains), (50 strains), 


Property positive positive 
strains strains 
Decomposition of : 
Casein 0 98 
Gelatin 36 100 
Tyrosine ) 100 
Xanthine 0 0) 
Hydrolysis of starch 58 56 
Acid from: 
Adonitol 0) 0 
Arabinose 0 0 
Erythritol 6 0 
Galactose 24 92 
Glucose 100 96 
Glycerol 9S 98 
Inositol 2 100 
Lactose 0 0) 
Maltose 6 ) 
Mannitol ) 94 
Mannose 19 68 
a-Methyl-p-glucoside ) 0 
Raffinose 0) 0 
Rhamnose 33 0 
Sorbitol 0 0 
Xylose ) 0) 
Nitrite from nitrate Sd 92 
Growth at: 
50°C 24 8) 
40°C 90, 56 
35°C 100 100 
28°C 100 100 
10°C 12 30 
Utilization of: 
Acetate 100 100 
Citrate 33° 98 
Malate 100 100 
Propionate 100 100 
Pyruvate 100 100 
Succinate 100 98 
Benzoate 0) 0) 


THE GENUS NOCARDIA 


TABLE 3 


Comparative properties of certain acid-fast nocardias (Suter, 1951) 


Aerobic 


. fastidiosa 
. leishmanii 
. caprae 


4 


. pretoriana 

. pulmonalis 

. paraffinae 

. transvalensis 


4 


4 


baths TE tis 


. polychromogenes 


4 


. minima 


4 


. coeliaca 


. rubropertincta 
. asteroides 


‘ 


. salmonicolor 


4 


rubra 
. farcinica 


b+++1+++++4+4+4+44 1 


4 


aie 98> | 


Pigment 
production 


;t++++t++4+4t4+ F441 


Growth at 
room temperature 


Aerial 
mycelium 


Growth on 
potato 


+++ 4+ 44444 
+++ 


_ 


Mycobacterium. In the case of the Strepto- 
myces-designated cultures, 83 per cent pro- 
duced sporulating aerial hyphae, 9 per cent 
nonsporulating aerial hyphae, and 8 per cent 
formed no aerial hyphae. The Nocardia- 
designated cultures gave, with regard to 
production of aerial hyphae, 24, 47, and 10 
per cent, respectively. Among the 214 Nocar- 
dia-designated cultures, 79 were recognized 
as representing N. (Eppinger) 
Blanchard. They all produced acid from glu- 
cose and glycerol, and utilized acetate, mal- 
ate, propionate, pyruvate, and succinate. 
They all grew well at 28 and 35°C, and 88 
per cent grew at 40°C. Eighty-six per cent 
reduced nitrate to nitrite, 54 per cent hy- 
drolyzed starch, and 34 per cent decomposed 
gelatin. A large number of cultures desig- 
nated as Nocardia (N. corallina, N. ery- 
thropolis, N. globerula, N. lutea, N. opaca, 
N. rhodni, N. rubra) were tentatively 
signed by Gordon and Mihm to the myco- 
bacteria under JW. rhodochrous (Overbeck) 


asteroides 


as- 


nov. comb. (Table 2). 

Of five species of aerobic actinomycetes 
associated with various mycetomas, Mariat 
(1957) recognized only N. asteroides and N. 
brasiliensis as nocardiae; Streptomyces ma- 


durae, S. pelletiert, and S. somaliensis were 
considered streptomycetes, although 
Mariat was not quite certain of their exact 
systematic position. 

Bojalil and Cerbon (1959) divided the 
genus Vocardia into two different metabolic 
groups: (1) Produces round colonies, adher- 
ing to wall and bottom of tube; utilizes 
gelatin as the only source of N and C, break- 
ing it down into amino acids and giving an 
alkaline reaction. NV. brasiliensis belongs to 
this group. (2) Produces flaky growth easily 


as 


dispersed through medium; poor growth on 
gelatin. N. asteroides belongs to this group. 

A detailed examination of the variability 
of different strains of two species of Nocar- 
dia with regard to their ability to utilize 
different 
well as in certain other physiological and 


‘carbon and nitrogen sources, as 


biochemical properties, is reported in Table 
2. Some comparative properties of several 
nocardiae are given in Table 3. 

Spalla (1958, 1959) eriticized the various 
descriptions of Nocardia species on the basis 
of an insufficient number of characters. He 
suggested that the following properties be 
used for characterization and classification 
of nocardiae: 


TABLE 4 


Cultural and biochemical properties of certain species of Nocardia (Spalla, 1959) 


Growth* on 


dese auiseied 
-Sse-asoonyyy 


VSR [OIIIA[H 


umtpaut 
auIWIe-7-N 


Other properties 


uint,aoAUL 
[euey 


a0uR\sIsal ploy 


Shae. 
jo uoTVR[NSRO) 


++t+4+ 01+ 


uljejas 
JO SISA[OIPAPHL 


++4++4+11+ 


yorrys 
Jo SISA[OIPAP{ 


ayei}iu 
jo_"uoTjONpay 


Acid production from: 


[Oq1oS-P 


jouuopy 


oat te oer ere abe atl 


jouuuryy-p 


++4+4+4 


JOIII AT) 


+++++4+4+4+ 


uypnuy 


Pecilee ae oR lh ele st 


asouyjey 


asoyeyatL, 


aso}IV'T 


9s0}[P 


asoiong 


asoulqeiy-/ 


+++4+111 


asouuem-p | | | | ++ I + 
ssoumvyy | +++ | + | 
S800 P |e ar ae oN Pl se 


asoyElRet)-p 


Organism 


. asterotdes........ 
blackwellit ....... 
DUPURIYRIR Pl cen 


Strain 959) 09224. 424 
Strain 2p. soe oe 


= 
= 
> 
S 
S 
= Zi Sk 


apricot; S = salmon. 


« 
c 


rose; L = lilac; A = 


colorless; R 


yellow; O = orange; C 


+ Now recognized as a Streptomyces. 


Fy 


THE GENUS 


NOCARDIA 29 


TABLE 5 


Serological relations of major pathogenic actinomycetes (Gonzdlez Ochoa and Vasquez Hoyos, 1953) 


Antigens 

N. brasilien- 1 

sis No. 447 
VAD ODUSINIOR Od lteeace ne, shetssertan Sk Shy bo ee + 
N. brasiliensis No. 447.............. + 
N. brasiliensis No. 468 + 
N. mexicana No. 414... ...........5.55: + 
INES OSTETOUACSRINO:, NSir. oe.ctescye oes oi0mrs, 6 a oes + 
INPASEENOUGES INO ANG). a ipttcaysc sauce cess + 
Nie astern ovdesiNOe20) .f0n5ecsb es ss a a + 
IN asteTvovdes NiOn O02b 6. 1355006050 +-00- + 
N. asteroides No. 694................... + 
INENGUPSOUEES NO. DOLL. oo. ek ete nates os + 
INEACOnNCG NO G16). 2. 5. es ws oe eh + 
N. leishmanii No. 1030............. + 


DSEMITLA UT LCUN Oma Diet. 1. 2 5 he tna: sie ee cise ts 
Se pelletventiNO., WISS.2 we... went ee _ 
SME PRUGANGUNO: GID. «ices icesicsue owes s = 
S. somaliensis No. 1065............... — 
SS; somalvensis No: 1064............-... = 
ISIS OMMALLENSUSMINO. LOGOR 5. cad a+ cle See ee - — 
S. paraguayensis No. 285.............. _ 
SPE OLOUISS NOM OU Siedanvaiee ss ur:.c sere hun ee ae — 
GHUSC US NO OhOy tty: bee idem aes syle as - 
im. wavenautae No: 9963... ......6.+:-:- — 


’. asteroides S. madurae 


Precipitin formation 


S. pelletiert S. somaliensis S. paraguay- 


No. 19 No. 412 No. 1185 No. 1065  ensts No. 285 
a — — — — 
aL == = = = 
_ — — — ae 
a = — — — 
aa — — — a 
a — — — = 
4. =— — — — 
+ — — — = 
aa = — — — 
a = = = = 
+ — — = = 
— — — = 
— — — + — 
== — — — — 
= - - + 


| 
| 
| 
| 
ap ct cea al 


1. Color of growth on several synthetic and 
at least one organic medium. 

2. Acid production from various carbon 
sources. 

3. Heat resistance. 

4. Size of terminal fragments. 

5. Staining properties, notably acid-fast- 
ness. 

6. Diastase formation. 

7. Gelatin liquefaction. 

8. Reduction of nitrate to nitrite. 

9. Coagulation and peptonization of milk. 

10. Formation of aerial mycelium. 

By utilizing these properties and consider- 
ing the high degree of similarity, Spalla was 
able to conclude that three mutants of NV. 
rugosa belonged to the same species as the 
parent, and that N. rugosa, N. 
rubra, and N. blackwellii represent distinct 
species, with a low degree of similarity. 


asteroides, N. 


A summary of some of the properties of 
N. rugosa and three of its mutants, as well 
as of certain other Nocardia species, is given 
in Table 4. The marked difference between 
the culture designated as NV. gardneri and the 
other nocardiae may be noted, particularly 
in utilization of carbon sources, starch hy- 
drolysis, and formation of aerial mycelium. 
These differences account for the fact that 
N. gardneri is now recognized as a Strepto- 
myces and not as a Nocardia. 

Various serological reactions of different 
species of Streptomyces and Nocardia are 
given in Table 5. Further information on the 
relation of Mycobacterium to Nocardia is 
found in the work of Haag (1927) and 
Gordon (1937). 

Of the 
Nocardia, the following three systems of 
classification of species are presented here: 


various treatments of the genus 


30 THE ACTINOMYCETES, Vol. II 


1. Classification of Nocardia* Species, 
According to Jensen (1932a) 


A. Partially acid-fast organisms with strongly re- 
fractive cells; nonproteolytic, generally non- 
diastatic; capable of utilizing paraffin. 

I. Initial mycelium limited, rapidly dividing 
into rods and cocci. 
1. Slowly growing organism; cells 0.5 to 

0.7 » in diameter. 

Nocardia minima 
2. Rapidly growing organisms; cells 1.0 to 

1.2 win diameter. 

a. Cystites not produced; rapid forma- 
tion of cocci. 

Nocardia corallina 
b. Cystites produced; less rapid forma- 
tion of cocci. 
Nocardia salmonicolor 
II. Initial mycelium well developed, richly 
branching, dividing into rods and generally 
into cocct. 
1. Substrate growth soft, without macro- 
scopically visible aerial mycelium. 

a. Substrate growth red; may produce 
variants with undivided substrate 
mycelium and visible white aerial 
mycelium, or yellow and white vari- 
ants. 

Nocardia polychromogenes 

b. Substrate growth white to pale pink. 
al. Growth in nutrient agar opaque, 


cream-colored; coeci in broth 
culture. 
Nocardia opaca 
b!. Growth on sugar-free nutrient 


agar watery; no cocci in broth 
culture. 
Nocardia erythropolis 
2. Substrate growth hard, yellow, with 
white aerial sporophores 
divide into chains of acid-fast cocci. 
Nocardia paraffinae 
B. Nonacid-fast organisms with weakly refractive 
cells; no distinct formation of cocci. Diastatic. 
I. Nonproteolytic; no aerial mycelium; 
marked production of cystites. 
Nocardia mesenterica 


mycelium ; 


Il. Proteolytic organisms. 

1. Growth on nutrient agar with rapid 
formation of unbranched diphtheroid- 
like rods; no typical cystites; broth tur- 
bid. 


Nocardia actinomorpha 


* Designated by Jensen as Proactinomyces. 


bo 


Growth with extensive mycelium on nu- 

trient agar; simple unbranched rods not 

formed; cystites present; broth clear. 
Nocardia flavescens 


2. Classification of Nocardia,* Accord- 
ing to Krassilnikoy 


A. Cultures colorless, some excreting a brown sub- 
stance into the medium. 
I. Aerial mycelium and spore-bearing hy- 
phae produced in culture media. 

1. Substrate and aerial mycelium ocea- 
sionally forming septae; the hyphae 
break up into long rods, 15 to 30 y; 
spherical bodies not formed. 

a. Saprophytes, found on dead sub- 
strates. 
Nocardia actinoides 
b. Parasites, living in bodies of man 
and animals. 
Nocardia gedanensis 

2. Mycelium producing frequent septae; 
hyphae break up into short rods and 
cocci. 

a. Saprophytes. 
al. Grow on protein media. 
Nocardia actinomorpha 
b!. Grow on paraffin. 
Nocardia paraffinae 
b. Parasites. 
Nocardia bovis 
Il. Cultures produce aerial sporophores on 
the surface of the colonies, but no aerial 
mycelium; sporophores are short, straight, 
covering the surface of the colonies with 
a thin, pale layer. 
1. Cultures grow in organic media. 
Nocardia albicans 


~o) 


2. Cultures grow in inorganic media. 
Nocardia oligocarbophilus 
III. Cultures not forming any sporophores or 
any aerial mycelium; colonies smooth or 
lichenoid. 
1. Saprophytes found on dead substrate. 
Nocardia albus 
2. Parasites or symbionts living within 
plants, animals, or man. 

a. Organisms living in symbiosis with 
plants, forming nodules on their 
roots. 

Nocardia alni 
Nocardia myricae 
Nocardia elaeagnii 


* Generic name Proactinomyces used. 


THE GENUS NOCARDIA 31 


b. Organisms living in the bodies of 


man and animals. 


al. Anaerobes, living in absence of 


oxygen. 
Nocardia anaerobicus 
b!. Aerobes, or  microaerophilic 
forms. 
a2. Strict aerobes. 
Nocardia lignierest 
b?. Facultative aerobes. 
a3’. Cells nonacid-fast. 
Nocardia israeli* 
b3. Cells acid-fast. 
Nocardia muris 
B. Cultures pigmented. 
I. Cultures pigmented violet or blue, the 
pigments diffusing into the substrate. 


1. Well 


produced, hyphae forming occasional 


developed substrate mycelium 
septae and breaking up into long rods, 
20 to 30 w; colonies form a faint aerial 
mycelium with straight sporophores; 
spores cylindrical. 

Nocardia gabritschewski 
2. No aerial mycelium produced, hyphae 
fre- 


or substrate mycelium forming 


quent and breaking up into 


short rods and cocci. 


septae 


Nocardia cyaneus 
Il. Red or orange pigment produced. 

1. Mycelium forming occasional septae 
and breaking up into long rods; some 
give rise to a faint aerial mycelium and 
short straight sporophores. 

a. Saprophytes. 
al. Cultures not forming any soluble 
pigment in medium. 
Nocardia fructifert 
b!. Cultures producing a brown sub- 
stance. 
Nocardia polychromogenes 
b. Parasites living in bodies of man and 
animals. 
Nocardia freerv 

2. Hyphae forming frequent septae and 

breaking up into short rods and cocci; 

no aerial mycelium produced. 

a. Saprophytes living on dead sub- 
strates. 

Nocardia ruber 

b. Parasites living in bodies of man 

and animals. 


* See Chapter 2 for description of Actinomyces 


israelit. 


FiGuRE 11. N. 
soil extract agar (Reproduced from: Gordon, R. E. 
and Mihm, J. M. J. Bacteriol. 75: 240, 1958). 


asteroides, strain 730, grown on 


al. Cells acid-fast. 
Nocardia asteroides 
bl. Cells nonacid-fast. 
Nocardia variabilis 
III. Cultures citron-yellow or bright yellow. 
1. Faint 
sporophores 


aerial mycelium with straight 
and cylindrical spores 
produced. 
a. Saprophytes. 
Nocardia flavescens 
b. Parasites. 
Nocardia somaliensis* 
2. No aerial mycelium produced. 
a. Saprophytes. 
al. Cultures yellow or bright yellow. 
Nocardia flavus 
bt. Cultures citron-yellow. 
Nocardia citreus 
b. Parasites. 
al. Cells acid-fast. 
Nocardia farcinica 
bl. Cells nonacid-fast. 
Nocardia putoriae 
IV. Cultures pigmented green. 
1. Saprophytes. 
Nocardia viridis 
2. Parasites. 
Noca rdia pyogenes 
V. Cultures black. 
1. Saprophytes. 
Nocardia nige | 
2. Parasites. 


Now a rdia Sé ndate ns 1s 


* Now recognized as a Streptomyces. 


32 THE ACTINOMYCETES, Vol. II 


3. Classification of Nocardia, According 
to the system of Waksman and 
Henrici* 


A. Partially acid-fast organisms with strongly re- 
fractive cells; nonproteolytic and generally 
nondiastatic; capable of utilizing paraffin. 

I. Initial mycelium fully developed, well 
branching, dividing into rods and generally 
into cocci. 

1. Substrate growth soft, without macro- 
scopically visible aerial mycelium. 
a. Substrate mycelium yellow, orange, 
or red. 
al. Pathogenic. 
a2. Substrate mycelium 
buff, or pale yellow. 
18. Nocardia farcinica 
b2. Substrate mycelium yellow to 
red. 
6. Nocardia asteroides 
b!. Not pathogenic. 
a2, Paraffin decomposed. 
42. Nocardia polychromo- 
genes 
b?. Cellulose decomposed. 
13. Nocardia cellulans 
b. Substrate mycelium white to pink. 
al. Gelatin not liquefied. 
a2. Growth on nutrient 
opaque, cream-colored. 
38. Nocardia opaca 
b?. Growth on nutrient agar pink. 
10. Nocardia calcarea 
a3, Aerial mycelium on milk 
white. 
31. Nocardia leishmanit 
b’. Pellicle on milk pink. 
11. Nocardia caprae 
e3, Pellicle on milk yellow. 
9. Nocardia brasiliensis 


white, 


agar 


d’. Causing galls on blueberry 
plants. 
53. Nocardia vaccinit 
b!. Gelatin liquefied. 
43. Nocardia pulmonalis 
2. Substrate mycelium hard, yellow. 
a. Aerial mycelium white; hyphae di- 
vides into chains of acid-fast cocci. 
40. Nocardia paraffinae 


* This system was used, with certain minor 
omissions and additions, in the last edition of 
Bergey’s Manual. 


b. Aerial mycelium not produced on or- 
ganic media. 

41. Nocardia petroleophila 
Substrate growth cream colored, later 
becoming yellow. 

51. Nocardia serophila 
Substrate growth hard, orange-yellow. 

58. Nocardia variabilis 


II. Initial mycelium very short, rapidly divid- 
ing into rods and cocci. 


a 


Growth pink. 
a. No cystites (swollen cells) formed. 
al. No indigotin from indole. 
16. Nocardia corallina 
b!. Indigotin from indole. 
26. Nocardia globerula 
b. Cystites formed. 
49. Nocardia salmonicolor 


. Growth coral-red. 


47. Nocardia rubropertincta 
Growth white, tan, or pink. 
a. No aerial mycelium. 
a!. Growth tan. 
15. Nocardia coeliaca 
b!. Growth white. 
28. Nocardia intracellularis 
b. Aerial mycelium produced. 
al. Growth frequently pinkish. 
53. Nocardia transvalensis 
b!. Growth never pink. 
50. Nocardia sebivorans 
Produces no pigment, no growth on 
potato, coagulates milk. 
19. Nocardia fastidiosa 


. Nonacid-fast organisms with weakly refrac- 


tive cells; no distinct formation of cocci; di- 


astatic. 


I. Nonproteolytic, although some give gela- 


tin liquefaction. 


i 


Growth on agar pale cream. 
a. Gelatin not liquefied; starch hy- 
drolyzed. 
35. Nocardia mesenterica 
b. Gelatin liquefied; starch not hy- 
drolyzed. 
48. Nocardia rugosa 
Growth on agar whitish. 
4. Nocardia albicans 
Growth on agar yellow. 
20. Nocardia flava 
Growth on agar green. 
59. Nocardia viridis 
Growth on agar yellow-green. 
14. Nocardia citrea 


6. 


10. 


14. 


Il. Proteolytic, 


. Growth 


THE GENUS 


Growth initially colorless, producing a 
yellow-green pigment in 2 to 4 days. 
54. Nocardia turbata 


. Growth on agar dark brown and even 


black. 

a. No liquefaction of gelatin. 
37. Nocardia nigra 

b. Gelatin liquefied. 
29. Nocardia tvorensis 


. Growth consistency soft; aerial my- 


celium sparse. 

33. Nocardia lutea 
consistency medium; aerial 
mycelium profuse. 

8. Nocardia blackwellit 
Growth cream-colored to pink; aerial 
spikes produced. 

52. Nocardia sumatrae 


. Growth grayish-yellow. 


36. Nocardia muris 


. Growth yellowish-orange. 


55. Nocardia uniformis 
Pigment on protein media deep brown. 
44. Nocardia rangoonensis 
Pigment on protein media light brown. 
12. Nocardia caviae 
although 


some are only 


weakly proteolytic. 


Ie 


Growth on nutrient agar with rapid 
formation of unbranched diphtheroid- 
like rods; no typical cystites; broth 
turbid. 

1. Nocardia actinomorpha 


. Growth white, shiny or pale. 


a. Dough-like consistency; breaks up 
into short rods. 
3. Nocardia alba 
b. Membranous, myceloid growth. 
32. Nocardia listert 
Growth on nutrient agar with extensive 
mycelium; simple unbranched rods not 
formed; cystites present. Broth clear. 
21. Nocardia flavescens 


. Growth cream-colored. 


a. Rapid liquefaction of gelatin. 
al. No aerial mycelium. 
25. Nocardia gibsonit 
bl. Aerial mycelium scant, white. 
56. Nocardia upcottit 
b. Slow liquefaction of gelatin. 
17. Nocardia dicksonii 


. Growth rose-colored to bright red or 


red-orange. 
24. Nocardia fructifera 


6. Growth pink to red. 


NOCARDIA 33 


FIGURE 12. N. 
produced from: McClung, N. M. First Reg. Conf. 
Asia and Oceania, Tokyo, 1956). 


rubra, electron micrograph (Re- 


a. Gelatin not liquefied. 
2. Nocardia africana 

b. Gelatin slowly liquefied or not at all. 
46. Nocardia rubra 

c. Rapid liquefaction of gelatin. 
39. Nocardia panjae 


7. Pigment on protein media light brown; 
color of growth pink. 
45. Nocardia rhodnii 
8. Growth yellowish to golden brown. 


22. Nocardia fordir 
9. Growth yellow to reddish-brown; solu- 
ble pigment brown to red. 
30. Nocardia kuroishi 
10. Growth tan to buff-colored. 
23. Nocardia formica 
11. Growth very limited on various media, 
except potato. 
27. Nocardia hortonensis 
12. Occur in the sea; liquefy agar. 
a. Growth yellow. 
34. Nocardia marina 
b. Growth yellow-orange. 
7. Nocardia atlantica 
13. Produce nodules on roots of plants. 
5. Nocardia alni 


In addition to the species included in the 


and described below, 


many more species of Nocardia have been 


above classification 
recorded in the literature, either under this 
or under other generic names. Some are 
listed in Chapter 13, under the incompletely 
described forms. Others are synonyms. There 
is no question that some of the Streptomyces 
species described in Chapter 8 could just as 


34 THE ACTINOMYCETES, Vol. II 


well have been included among the Nocardia 
forms. It is also possible some of those listed 
as Nocardia could just as readily have been 
included in the genus Streptomyces. Fre- 
quently, the decision of the investigator as 
to whether a certain culture should be in- 
cluded in one genus or another was perfectly 
arbitrary. 


Descriptions of Nocardia Species* 


1. Nocardia actinomorpha (Gray and 
Thornton, 1928) Waksman and Henrici, 
1948 (Gray, P. and Thornton, H. Centrl. 
Bacteriol. Abt. II, 73: 88, 1928). 

Morphology: Growth colorless, smooth, 
consisting of long, branching filaments and 
rods, 0.5 to 0.8 by up to 10 yw. In older cul- 
tures, rods 2 to 3 uw long generally predomi- 
nate. On some media, extensively branching 
hyphae occur. Not acid-fast. 

Nutrient agar: Round colonies, 1 mm in 
diameter, convex, white, granular or resin- 
ous; long arborescent processes from the 
edge. No aerial mycelium. 

Potato-glycerol agar: 
wrinkled, pink to orange. 

Egg medium: Growth raised, dry, smooth, 


Growth dry, 


salmon-buff. 
Gelatin: Colonies round, 
white, raised rim, edges burred. Liquefaction 


saucer-like, 


positive. 

Nutrient broth: Turbid. 

Milk: Coagulation and peptonization. 

Starch: Hydrolyzed (diastase produced). 

Sucrose: Inverted. 

Nitrate reduction: Positive. 

Phenol and naphthalene: Utilized. 

Temperature: Optimum 25-30°C. 

Source: Soil. 

Remarks: Differs from N. 
liquefaction of gelatin. No acid from glucose, 
lactose, sucrose, or glycerol. 


coeliaca in 


2. Nocardia africana Pijper and Pullinger, 


* For further details concerning some of the 
species, the last edition of the Bergey Manual 


should be consulted. 


1927 “(Piper “As-andPullinger,-B. DD. 23: 
Trop. Med. Hyg. 30: 153-156, 1927). 

Synonym: Actinomyces africanus (Pijper 
and Pullinger) Nannizzi Pollaceci, 1934. 

Morphology: Substrate growth consists of 
unicellular branching mycelium. Aerial my- 
celium sparse, consisting of short, straight 
hyphae. Not acid-fast. 

Glucose agar: Colonies minute, red, dis- 
crete, round and piled up into a pale pink 
mass. Aerial mycelium thin, white. 

Nutrient agar: Colonies discoid, flat, pink. 

Glycerol agar: Growth made up of small, 
heaped-up, colorless masses with pink tinge; 
later, growth abundant, piled up, pale pink. 

Potato agar: Growth bright red, made up 
of small, round colonies with colorless sub- 
merged margins, and piled up patches. Aerial 
mycelium stiff, sparse, white. 

“ge medium: Colonies small, colorless, 
blister, partly confluent ; becoming wrinkled, 
depressed into medium. Liquefaction shght. 

Gelatin: Irregular pink flakes. No liquefac- 
tion. 

Milk: Surface growth bright red. Medium 
gradually becomes opaque, reddish-purple, 
with slow peptonization. 

Source: A case of mycetoma in South 
Africa. 

3. Nocardia alba (Kxrassilnikov, 1941) 
Waksman (Krassilnikov, N. A. Actinomyce- 
tales. Izvest. Akad. Nauk. SSSR, Moskau, 
1941, p. 1). 

Morphology: Growth smooth or folded, 
made up of white colonies of a dough-lke 
consistency; shiny or pale. Substrate myce- 
lium breaks up into short rods 2.7 by 0.7 to 
0.8 uw, later changing into a mass of coccus- 
like cells, 0.7 to 1 uw. Many cells are swollen, 
others form side buds. Not acid-fast. No 
aerial mycelium. 

Synthetic agar: 
sources of nitrogen; sugar, starch, or organic 


Inorganic salts used as 


acids utilized as sources of carbon. 
Nutrient agar: Good growth. No aerial 
mycelium. 


THE GENUS 


Gelatin: Growth good. Positive liquefac- 
tion. 

Milk: Coagulation and peptonization. 

Starch: Rapid hydrolysis. 

Cellulose: No growth. 

Paraffin: No growth. 

Nitrate reduction: Negative. 

Sucrose: Inverted. 

Source: Soil. 

Remarks: Several subspecies were also 
listed: N. chromogena, N. paulotropha (Ac- 
tinobacillus paulotrophus Beijerinck, 1914), 
N. alba lactica, N. diastatica, N. hoffmanni. 


4. Nocardia albicans (Krassilnikov, 1941) 
Waksman (Krassilnikov, N. A. Actinomyce- 
tales. Izvest. Akad. Nauk, SSSR, Moskau, 
1941). 

Morphology: Growth red, hyphae break- 
ing up into rod-shaped cells, 12 to 25 by 0.6 
to 0.7 uw, up to 50 uw in length. Cells straight 
or slightly curved, branching. Aerial myce- 
lium not observed, except surface layer of 
sporophores, which produce a velvety ap- 
pearance. Multiplication by fission, seldom 
by budding. 

Nutrient 
shiny. 

Gelatin: No liquefaction. 

Milk: No change. 

Starch: Hydrolyzed. 

Cellulose: No growth. 

Nutrient broth: Growth poor; produces 


agar: Growth good, smooth, 


faint turbidity, which settles on bottom and 
leaves a surface ring. No true mycelium. 
Cells rod-shaped 5 to 10 uw, seldom 15 to 20 u. 

Nitrate reduction: Negative. 

Sucrose: Inverted. 

Paraffin: Not utilized. 

Source: Soil. 

Remarks: Glycerol used as a 
‘arbon, and nitrate as a source of nitrogen. 


5. Nocardia alni (Peklo emend. v. Plotho, 


1941) Waksman (von Plotho, O. Arch. 
Mikrobiol. 12: 1-18, 1941). 
Morphology: Mycelium contains fatty 


NOCARDIA 


source of 


ey) 
~ 


globules; cells filiform, branching, disinte- 
Aerial 


mycelium usually absent, but may be formed 


grating into short rods and cocci. 
on cultivation. Sporulating cultures form 
white, spherical to oval spores. 

Agar media: Substrate growth compact, 
shiny, colorless or slightly brownish. 

Gelatin: pellicle. 
positive. Soluble pigment brownish. 

Liquid media: Slimy surface film. 

Tyrosine: Utilized as source of nitrogen; 
color turns red-brown. 

Cellulose: Not utilized. 

Carbon sources: Produces lactic acid from 


Surface Liquefaction 


glucose and lactose. 

Optimum reaction for growth: pH 6.0. 

Habitat: Roots of the alder. 

Remarks: Produces nodules on the roots 
of the host plant. Said to bring about nitro- 
gen fixation in symbiotic culture with the 
plant. 


6. Nocardia asteroides (Kppinger, 1891) 
Blanchard, 1895 emend. Gordon and Mihm, 
1959 (Eppinger, H. L. Beitr. Pathol. Anat. 
9: 287, 1891; Blanchard, R. In Bouchard. 
Traite Pathol. Gen. 2: 811, 1895; Gordon, 
R. E. and Mihm, J. M. J. Gen. Microbiol. 
20: 129, 1959). 

Synonyms: Cladothrix asteroides Mppinger, 
1890; Streptothrix eppingeri 
1891; Actinomyces asteroides Gasperini, 1892; 


Lossi- Doria, 


Oospora asteroides Sauvageau and Radais, 
1892; N. asteroides R. Blanchard, 1895. Ac- 
cording to Ochoa and Sandoval (1956), N. 
leishmanii. Chalmers and Christopherson, 
and N. phenotolerans Werkam and Gammel 
are synonyms of NV. asteroides. According to 
Gordon and Mihm (1959), V. caprae (Silber- 
schmidt) Waksman Henrici, N. ep- 
pingert, N. minima, and N. sylvodorifera are 
also synonyms. 

Morphology : 
growth, usually yellow to orange to orange- 
red. Mycelium straight and fine; it breaks up 
into small, coceoid forms and rods. Some 


and 


Typical actinomycete 


strains are acid-fast ; others are only partially 


36 THE ACTINOMYCETES, Vol. II 


so. Aerial hyphae produced; they vary from 
rudimentary to long branching. Some may 
produce chains of spores (Fig. 11). 

Sucrose nitrate agar: Growth thin, spread- 
ing, orange. No aerial mycelium. No soluble 
pigment. 

Peptone-beef extract agar: Growth much 
folded, light yellow, becoming deep yellow 
to yellowish-red. No soluble pigment. 

Yeast-glucose agar: Growth flat to 
wrinkled, beige to dark pink. Some produce 
white aerial hyphae. 

Potato: Growth much wrinkled, whitish, 
becoming yellow to almost brick-red. 

Gelatin: Growth yellowish on surface. No 
liquefaction. 

Milk: Orange-colored ring. No coagula- 
tion; no peptonization. 

Starch agar: Growth restricted, 
orange. No diastatic action. 

Blood serum: No liquefaction. 

Carbohydrate utilization: See Table 2. 

Nitrate reduction: Positive. 

Oxygen demand: Aerobic. According to 
Chalmers and Christopherson (1916), it may 
also grow anaerobically. 

Temperature: Optimum = 37°C. 
strains grow readily at 28°C. 

Pathogenicity: Transmissible to rabbits 
and guinea pigs, but not to mice. 

Source: Human infections and soil. 

Remarks: A number of strains of acid-fast 


scant, 


Some 


actinomycetes isolated from human lesions 
have deviated in certain particulars from 
the description of N. asteroides, but not 
sufficiently to warrant separation as different 
species. According to Gordon and Mihm, all 
strains of N. asteroides form whitish aerial 
hyphae, these varying from rudimentary to 
much branching. The following characteris- 
tics were considered the most valuable in the 
identification of the species: development of 
filamentous colonies with aerial hyphae; fail- 
ure to hydrolyze casein and to dissolve the 
crystals of tyrosine and xanthine; acid pro- 
duction from glucose and glycerol; failure to 


form acid from arabinose, lactose, mannitol, 
inositol, and xylose; utilization of acetate, 
malate, propionate, pyruvate, and succinate, 
but not benzoate. 

Numerous varieties of this species have 
been described. It is sufficient to mention 
N. crateriformis, N. gypsoides, and N. pseudo- 
carneus (Gordon and Mihm, 1957). 

Type culture: IMRU* 3308; also 504. 


7. Nocardia atlantica (Humm and Shep- 
ard, 1946) Waksman (Humm, H. J. and 
Shepard, K.S. Duke Univ. Marine Sta. Bull. 
3: 78, 1946). 

Synonym: Proactinomyces atlanticus. 

Morphology: Hyphae long, branching, 
breaking up into rods and cocci, 0.5 to 0.7 pu. 
Involution forms in old cultures. Nonacid- 
fast. Aerial mycelium not produced. 

Synthetic and organic media: Growth 
bright yellow or yellow-orange, smooth, com- 
pact, of a doughy consistency. Colonies flat 
with shghtly raised center. Soluble pigments 
none. Mineral sources of nitrogen utilized. 

Gelatin liquefaction: Positive. 

Agar: Liquefied. 

Milk: Coagulation rapid, acidified; pep- 
tonization slow. 

Nitrate reduction: Positive. 

Starch: Hydrolyzed. 

Cellulose: Decomposed. 

Chitin: Decomposed. 

Agar: Slowly digested. 

Alginie acid: Decomposed. 

Carbon sources: Acid produced from arab- 
inose, xylose, rhamnose, raffinose, fructose, 
galactose, gum arabic. No acid 
from lactose, dulcitol, mannitol, or sorbitol. 
Organic acids utilized: gluconie, lactic, malo- 
not utilized: acetic, 


sucrose, 


nic. Organie acids 
butyric, citric, ete. 

Optimum temperature: 28-30°C. 

Habitat: Marine algae and marine sedi- 
ments. 

* These designations represent the various cul- 
ture collections where the type cultures are de- 
posited. 


THE GENUS 


Remarks: Another closely related culture 
has been described as Proactinomyces flavus 


(see N. marina). 


Nocardia blackwellii (Erikson, 1935) 
Waksman and Henrici, 1948 (Erikson, D. 
aa ee irch Council Spec. Rept. Ser. 203: 

, 1935). 
es 
rod-like filaments, 
sparsely branching hyphae. Aerial mycelium 

short, straight; frequently large, round 
ovoid cells are interposed in the irregularly 


Growth consisting of short, 


growing out into longer 


segmented chains of cells. 
Glycerol nitrate agar: 
granular, irregular, thin, pinkish. 


Growth extensive, 


Nutrient agar: Growth confluent, wrin- 
kled, with small, round, pinkish, discrete 


colonies at margin. 

Glucose nutrient agar: Growth abundant, 
pale pink, in form of small conical colonies, 
piled up, convoluted. 

Potato agar: Colonies small, round, color- 
less. Aerial mycelium white. Later, colonies 
dull pink, submerged margins; few aerial 
spikes, moderate aerial mycelium at top of 
slant. 

Gelatin: Colonies few, 
along line of inoculation. 
colorless colonies to 10 mm_ below 
larger pink-yellow surface colonies 
white aerial mycelium. No liquefaction. 

Milk: Surface pellicle heavy, convoluted, 
bright yellow. No coagulation; no peptoniza- 
tion. Spalla states that milk is coagulated. 

Carbon utilization: See Table 4 

Source: Hock joint of foal. 

Type culture: ATCC 6846; 


colorless, minute, 
Later, abundant, 
surface; 
with 


NCTC 630 


9. Nocardia brasiliensis (Lindenberg, 1909) 
Castellani and Chalmers emend. Gordon and 
Mihm, 1959 (Lindenberg, A. Arch. Parasitol. 
13: 265-282, 1909; Castellani, A. and Chal- 
mers, A. J. Manual of Tropical Medicine, 2d 


Kd. William Wood & Co., 1913, p. 816; 
Gordon, R. E. and Mihm, J.M. J. Gen. 
Microbiol. 20: 129, 1959). 


NOCARDIA 37 


Synonyms: Gonzalez Ochoa (1945, 1953), 
Gonzalez Ochoa and Sandoval ee and 
Gordon and Mihm (1958) consider bra- 


siliensis as the proper name for ae organ- 
ism. <A. Boyd and Crutchfield, 
N. pretoriana Pijper and Pullinger, f 


mMextCanus 
and 
transvalensis Pijper and Pullinger, are con- 
sidered as synonyms. 
Morphology: Angularly 


branched _ fila- 


ments, bearing a few short straight aerial 
hyphae; later, growth becomes spreading 
and extensive. Aerial hyphae long and 


branching to short and gnarled; divide to 
form oval and cylindrical spores. Acid-fast- 
ness variable, from 100 per cent to none. 

Glycerol nitrate agar: Growth in form of 
piled up pink mass. Aerial mycelium very 
secant, white, at margin. 

Glucose nutrient agar: Colonies pale buff, 
umbilicated and piled up. 

Yeast-glucose agar: Highly mutable. 
Growth yellow to yellowish-orange, finely 
wrinkled. Some strains produce no aerial 
hyphae; other strains form mat of whitish 
aerial hyphae. A few strains form amber to 
brown soluble pigment. 

Potato: Colonies small, raised, pale pink; 
plug and liquid discolored. Later, growth 
dull buff, dry and convoluted at base, round 
and zonate at top of slant. Aerial mycelium 
white. 

Gelatin: 
faction 

Milk: Surface growth yellowish. Pale pink 
growth up the wall of the tube. Solid coagu- 
lum in | month; later, partly digested. 
round, 
raised pink 


A few colorless flakes. No lque- 


‘ge medium: Colonies few, color- 


less in 3 days. Later, irregular, 
mass, warted appearance; moderate degree of 
liquefaction. 

convoluted, 


Serum Growth raised, 


slightly pinkish. 


agar: 
Source: A case of mycetoma of the chest 
wall in a South African native. 

To guinea pigs and humans. 
and 


Pathogenicity: 


Remarks: According to Gordon 


38 THE ACTINOMYCETES, Vol. II 


Mihm, NV. brasiliensis is distinguished from 
N. asteroides by positive decomposition of 
vasein and tyrosine and by acid formation 
from inositol and mannitol. Additional char- 
acteristics of this species include the follow- 
ing: xanthine not decomposed; acid pro- 
duced from glucose and glycerol; no acid 
from arabinose, lactose, maltose, xylose, and 
other sugars; utilizes acetate, citrate, malate, 
propionate, pyruvate, and succinate, but not 
benzoate. 

According to Mariat (1958), N. 
is characterized by a lack of proteolytic ac- 
tivity; utilization of urea, (NH4)oSOs and 
IXNO; as sources of nitrogen, not of nitrite. 
Glucose, fructose, glycerol, and mannitol are 


asteroides 


utilized as sources of carbon, but not galac- 
tose, xylose, maltose, and starch, although 
paraffin is utilized. 

N. brasiliensis is characterized by gelatin 
hydrolysis; utilization of urea, (NH4)2SO,4 
and KNO; as nitrogen sources; utilization 
of glycerol, glucose, fructose, galactose, man- 
nitol, xylose, and paraffin as carbon sources. 

Type culture: IMRU 850. 


10. Nocardia calcarea Metealfe and 
Brown, 1957 (Metealfe, G. and Brown, M. 
E. J. Gen. Microbiol. 17: 568-569, 1957). 

Morphology: Gram-positive and partially 
acid-fast. Mainly short rods (1.5 to 2.0 by 
1.0 uw) together with unbranched aseptate 
filaments up to 10 win length and occasional 
branched filaments. filaments show 
lemon-shaped swellings. After 4 days, short 
rods snapping division typical of 
corynebacteria; abundant unbranched fila- 
ments (5.0 to8.0 by 1.0 w) and a few branched 
filaments and cocci (1.0 uw) also present. 
Many rods show differentiation of a swollen 


Some 


show 


spore-like structure; these are usually formed 
terminally or subterminally, one per rod. In 
the filaments they are often formed in chains. 
Colonies after 14 days consist of short rods, 
cocel and rods with swollen cells. 

Agar media: Colonies circular, raised, soft, 
cream 


without aerial mycelium; pink or 


colored with distinct pink tinge when small. 


Sucrose agar: Filaments are rare and short 
rods and cocci are the predominant forms 
throughout. Occasional lemon-shaped cells 
are formed on all media. 

Glucose and mannitol agars: Very long 
branched filaments (10 to 25 w) present after 
2 days, often with terminal chains of swollen 
hyaline cells; these filaments usually frag- 
ment, but a few persist. 

Yeast extract-peptone agar: Cycle shorter 
than on previous media, most of the fila- 
ments having fragmented into rods and cocei 
after 3 days. 

Starch agar: Growth slight; no hydrolysis. 

Milk: Heavy growth, turned alkaline; no 
peptonization. 

Gelatin: Beaded growth at top of stab. 
No liquefaction. 

Nitrate reduction: Positive. 

Carbon utilization: Utilizes glucose, su- 
crose, and maltose; poor growth with lactose. 

Paraffin: Growth heavy in basal salts me- 
dium with ammonium salt and flakes of 
paraffin wax. 


11. Nocardia caprae (Silberschmidt, 1899) 
Waksman and Henrici, 1948 (Silberschmidt, 
W. Ann. inst. Pasteur 13: 841-853, 1899). 

Synonyms: This organism has been var- 
iously described as S. caprae (Price-Jones, 
1901). O. caprae (Sartory, 1923), A. caprae 
(Nannizzi, 1934). 

Morphology: Substrate growth forms thin, 
branching filaments, breaking up into rods. 
Aerial mycelium abundant on all media with 
tendency to form coherent spikes; mycelium 
very polymorphous, but 
thicker segments appear. Slightly acid-fast. 


not occasional 
Brownish soluble substance. 

Glucose-peptone-beef — extract 
Growth irregular, bright pink, tending to be 


agar : 


heaped up. Later abundant masses, frosted 
over with thin, white aerial mycelium. 

Blood agar: Colonies minute, round, color- 
less, aggregated in broad pink zones. Aerial 
mycelium pale. No hemolysis. 

Ege medium: Colonies tew, colorless, some 
pink; aerial mycelium white. Later, growth 


becoming dull pink, irregular, with scant 
white aerial mycelium. 

Potato: Growth abundant. Aerial myce- 
lium pale pink. Growth becomes membra- 
nous, considerably buckled. 

Gelatin: Growth extensive, dull, 
small raised patches of pink aerial mycelium; 
later, ribbon-like, depressed. No liquefac- 


with 


tion. 

Milk: Surface pellicle red. Solid coagulum; 
no peptonization. 

Starch: No hydrolysis. 

Source: Lesions in goats. 

Pathogenicity: To rabbits, guinea pigs, 
and mice. 

Remarks: According to Gordon and Mihm 
(1959) N. caprae is a synonym of N. aste- 
roides; according to Schneidau and Shaffer 
(1957), however, the organism does not uti- 
lize paraffin and does not hemolyze blood, 
as shown on p. 68, Volume I. 

Type culture: IMRU 783. 

12. Nocardia caviae Snijders, 1924 (Snij- 
ders, Geneesk. Tijdschr. Ned. Indie 64: 47, 
75, 1924). 

Morphology: Growth consists of initial 
segmented hyphae, producing elements of 
approximately even thickness, arranged in 
angular apposition; later, forms long, pro- 
fusely ramifying threads with strongly re- 
fractile protoplasm. Aerial mycelium straight 
and branching, the sporophores forming oc- 
casional coiled tips, divided into eylindrical 
spores. 

Glucose agar: Growth piled up, convo- 
luted, cream-colored to pale pink. Aerial 
mycelium white. 

Glycerol agar: Growth scanty. 

Potato agar: Growth spreading, colorless. 
Aerial mycelium dense white. 

Egg medium: Growth heavily corrugated, 
pale pink, with submerged margin. Aerial 
mycelium dense white. After 3 weeks, color- 
less transpired drops. 

Potato: Colonies small, colorless. Aerial 
mycelium white, powdery. Later, abundant, 


THE GENUS NOCARDIA 3 


raised, pale pink, confluent growth. Aerial 
mycelium white. Plug discolored. 

Gelatin: A few colorless flakes. No lique- 
faction. 

Milk: Surface growth colorless. Aerial 
mycelium white. Coagulation positive. 

Nutrient broth: Surface pellicle cream- 
colored, wrinkled, extending up wall and 
breaking easily; moderate bottom growth, 
flaky. Medium discolored. 

Source: Infected guinea pigs from Su- 
matra. 

Remarks: Schneidau and Shaffer (1957) 
report that the organism is not acid-fast, 
grows at 46°C, utilizes paraffin, liquefies 
gelatin, hydrolyzes casein, liquefies starch, 
and shows positive hemolysis. 


13. Nocardia cellulans Metealfe and 
Brown, 1957 (Metealfe, G. and Brown, M. 
E. J. Gen. Microbiol. 17: 569-570, 1957). 

Morphology: Gram-positive and partially 
acid-fast. Branching aseptate filaments, 30 
to 40 uw in length, often with swellings at in- 
tervals; shorter filaments are less than 7.0 u 
in length. Fragmentation commences about 
the fourth day, the number of short rods 
(1.56 to 2.0 by 1.0 uw) increasing rapidly. 
Hyaline spore-like structures are produced 
from the seventh day as slight terminal 
swellings on the filaments. After 28 days, the 
colonies consist of very short rods, cocci and 
spore-like cells. 

Agar media: Colonies raised, soft, without 
aerial mycelium; cream-colored on most 
media but characteristically bright yellow on 
yeast extract-peptone agar. 
agar: Filaments fragment 
rapidly and are occasionally found after 28 
days. Numerous Y-forms are found in older 


less 


Glucose 


cultures. 

Cellulose tubes: After 6 days there are 
long (20 to 30 uw) branched and unbranched 
with swellings. 


filaments, terminal 


Fragmentation is rapid and short rods and 


many 


cocci predominate during the stage of active 
cellulose decomposition. Old cultures are 
composed almost entirely of cocci. 


40 THE ACTINOMYCETES, Vol. II 


Milk: Acid and curd produced. 

Gelatin: Beaded growth at top of stab. 
No liquefaction. 

Starch agar: Starch not hydrolyzed. 

Nitrate reduction: Positive. 

Carbon utilization: Glucose, sucrose and 
maltose utilized; acid produced. 

Paraffin: Growth heavy with trace of 
yeast extract; no growth without yeast ex- 
tract. 

Type culture: ATCC 12,830. 


14. Nocardia citrea (IKXrassilnikov, 1938) 
Waksman and Henrici, 1948 (Krassilnikov, 


N. A. Bull. Acad. Sci. USSR No. 1: 139, 
1938). 
Morphology: Growth yellow to yellow- 


green, usually rough and folded, of a dough- 
like consistency. No soluble pigment. In 
young cultures, mycelium consists of very 
very fine threads 0.3 to 0.5 mw in diameter. 
After several days the cells break up into 
short rods 0.5 by 1.5 to 5 w and into cocci 
0.3 to 0.5 w in diameter. Cells are the small- 
est of all the nocardias. Multiplies by fission 
and bud formation. No aerial mycelium. Not 
acid-fast. 

Synthetic medium: Growth and pigmenta- 
tion typical. 

Nutrient agar: Growth good. 

Gelatin: Liquefaction rapid. 

Milk: Coagulation and peptonization. 

Starch: Hydrolyzed rapidly. 

Sucrose inversion: Positive. 

Cellulose: No growth. 

Mat: Weak growth. 

Paraffin or wax: No growth. 

Nitrate reduction: Positive. 

Habitat: Soil and water. 


15. Nocardia coeliaca (Gray and Thornton, 
1928) Waksman and Henrici, 1948 (Gray, 
P., and Thornton, H. Centr. Bakteriol. 
Parasitenk. Abt. II, 73: 88, 1928). 

Morphology: Growth in form of short, 
curved, uneven-sided rods, 0.8 by 5 u; oc- 
casional filaments up to 10 to 12 yp long; fre- 


quently beaded, occasionally swollen or 
branched; coecoid forms 0.8 to 1.2 w in 
diameter are common, especially in older 
cultures. Not acid-fast, or 
shghtly acid-fast. 

Nutrient agar: Colonies less than 1 mm 
in diameter, round or irregular, raised, white, 
resinous, edge irregular, burred. Deep col- 
ones irregularly round or oval, edge slightly 
broken. Slant filiform, convex, white, rugose, 
resinous, edge undulate. 


occasionally 


Potato-glycerol agar: Growth dry, crum- 
pled, orange-colored, becoming brown. 

Gelatin: Surface colonies irregular, raised, 
white, rugose, dull edge entire. Deep colonies 
irregular, smooth or slightly broken. Stab 
convoluted, buff-white to yellowish, dull. 
Below surface the growth forms many ir- 
regular hollow lobes, giving a glistening ap- 
pearance, to a depth of 3 to 4 mm. 

Milk: Shghtly alkaline after 5 to 7 days. 

Nutrient broth: Turbid. 

Nitrate reduction: None. 

Phenol: Utilized. 

‘ge medium: Growth 
moist, verrucose, buff-colored. 

Temperature: Optimum 22-25°C. 

Source: Soil. 

Remarks: No acid from glucose, lactose, 
sucrose, or glycerol. No  chromogenesis. 
Hollow lobes produced in deep gelatin cul- 


raised, smooth, 


tures. 

Type culture: ATCC 13181. 

16. Nocardia corallina (Bergey et al., 1923) 
Waksman and Henrici, 1948 (Hefferan, M. 
Centr. Bakteriol. Parasitenk. Abt. II, 11: 
459, 1904; Bergey et al., Manual, Ist ed., 
1923, p. 93). 

Synonyms: Nocardia minima (Proactino- 
myces minimus Jensen). Bacillus mycoides 
corallinus Reader, 1926. 

Morphology: Growth pink to 
orange-yellow. Branching mycelium, gener- 
ally curved. In older cultures, hyphae de- 
generate generally into shorter rods and 


red to 


coeel. Not acid-fast. 


THE GENU 


Nutrient agar: Colonies smooth, pink, 
shining; border lighter, edge filamentous or 
with arborescent projections. As the colony 
grows, the cells in the interior break up into 
short rods and cocci which eventually form 
the mass of the colony. Cells on the outside 
remain filamentous, giving the colony a 
burr-like appearance, and often forming long 
arborescent processes. No soluble pigment. 
Potato-glycerol agar: Growth filiform, 
raised, dry, wrinkled, yellowish-brown to 
coral red. 

Gelatin: Surface colonies round, convex, 
smooth, pink, shining, edge filamentous; 
deep colonies, burrs. No liquefaction. 

Milk: Reddish pellicle; milk becomes alka- 
line. 

Nutrient 
scum. 

Paraffin and phenol: Utilized. 

Nitrate reduction: Positive. 

Starch: Not decomposed. 

Sucrose: Not inverted. 

Kee medium: Filiform, 
wrinkled, orange. 

Temperature: Optimum 22-25°C. 

Habitat: Soil. 

Remarks: Some strains produce acid from 
glycerol and glucose. No acid or gas from 


broth: Usually turbid. Pink 


raised, dry, 


sucrose, maltose, or lactose. Phenol and m- 
cresol are utilized. strains utilize 
naphthalene. Krassilnikov (1949) reports for 
his strains, good growth in high salt concen- 
trations. Schneidau and Shaffer (1957) report 


Some 


positive acid-fastness, positive hemolysis 


and urease formation. 
1935) 


Research 


17. Nocardia dicksonii (Trikson, 
Waksman (Erikson, D. Med. 
Council Spec. Rept. Ser. 17: 203, 1935). 

Morphology: Growth consists of long fila- 
ments, sometimes wavy. Aerial mycelium 
straight. Spores cylindrical. 

Glycerol nitrate agar: Growth granular 
and wrinkled, cream-colored. Medium deeply 
discolored. 


NOCARDIA 41 


Glucose-asparagine agar: Growth wrin- 
kled, colorless. 

Potato agar: Growth abundant, colorless. 

Ege medium: Growth yellowish-brown. 

Starch agar: Strong hydrolysis. 

Gelatin: Growth smooth, cream-colored on 
surface. Liquefaction limited. 

Milk: Coagulated, peptonized. 

Habitat: Unknown. 


18. Nocardia farcinica (Nocard, 1888) 
Trevisan and De Toni, 1889 (Noeard, M. 
E. Ann. inst. Pasteur, 2: 293, 1888: Trevi- 
san, V., I. generi e le specie delle Batteri- 
acee, Milan, 1889, p. 9). 

Morphology: Growth yellow, of doughy 
consistency. Markedly acid-fast. 

Nutrient agar: Colonies yellowish-white, 
irregular, refractive; mycelium filamentous. 

Potato: Growth abundant, dull, crumpled, 
whitish-yellow. 

Gelatin: Colonies small, circular, trans- 
parent, glistening. No liquefaction. 

Milk: No coagulation; no peptonization. 

Starch: No hydrolysis. 

Nutrient broth: Clear, with granular sedi- 
ment, often with gray pellicle. 

Nitrate reduction: Negative. 

Temperature: Optimum 37°C. 

Pathogenicity : 
domestic animals and guinea pigs. 

Source: Cases of cattle farcy. 

Type culture: IMRU 3318. 


Pathogenic to certain 


19. Nocardia fastidiosa Suter, 1951 (Suter, 
L. S. Mycologia 43: 658-676, 1951). 

Morphology: The organisms were similar 
on all media studied, showing in general a 
striking pleomorphism with coccoid, bacil- 
lary, and filamentous forms. Many of these 
were clubbed; some bore a striking resem- 
blance to spermatozoa. Others were thick at 
one end and tapered down to filamentous 
tails. Filamentous forms were up to 25 uw in 
length and, not considering clubs or swollen 
portions, measured 0.2 to 1.2 u in diameter, 
the average being about 0.8 to 1 uw. Stained 


42 THE ACTINOMYCETES, Vol. II 


preparations never showed a richly branch- 
ing character, but an alternate type of 
branching was fairly easy to demonstrate 
after about 7 days’ incubation at 37°C. The 
eoccoid forms were round, oval, or drop- 
shaped. Neither septa nor nuclei were seen. 
Spores were formed in short chains within 
mycelial strands and were of the same diame- 
ter as the mycelial strands. Similar spores 
were also found singly and extracellularly. 
The organisms taken from cultures were 
partially acid-fast. 

Growth on agar media: Colonies were slow 
growing, appearing after 2 to 3 days as tiny 
specks, which after 7 days’ incubation finally 
achieved, but never exceeded, a size of about 
1 mm in diameter. To the naked eye they 
appeared = grayish-white, compact, and 
smooth, and under low-power magnification 
they appeared fluffy, raised, compact at the 
center, and irregular and stringy at the edge, 
due to the presence of radiating and tangled 
filaments. Zigzag arrangements of elements 
and branches, clubs, and curls were seen at 
the periphery. The colonies were adherent 
to the medium. The top surface was dry and 
could be scraped off with a stiff wire loop, 
but neither the whole colony nor any part 
of it could be removed intact. On blood agar 
after 7 days’ incubation, the colonies viewed 
by transmitted light showed a characteristic 
dense reddish center and a clear outer zone, 
both areas being very sharply defined. 

Optimum temperature: 37°C. 

Oxygen requirements: The organism is a 
facultative anaerobe, growing equally well in 
the presence or absence of oxygen. 

Proteolytic activity: The organism is non- 
proteolytic. No odor of putrefaction was 
perceived in any of the cultures; gelatin was 
not liquefied; no growth occurred on serum 
plates or on coagulated human serum. 

Gelatin stab: No growth after 28 days at 
17-20°C. 

Potato: No growth at 37°C. 

Carbon sources: Acid formed from glucose; 


not from lactose, sucrose, maltose, or glye- 
erol. 

Nitrate reduction: Negative. 

Habitat: Isolated from penile ulcer. 

Remarks: N. fastidiosa is different from 
previously described species of Nocardia in 
the following ways: It is very fastidious in 
its growth requirements. It does not grow 
in synthetic media to which carbohydrates 
have been added; it will not utilize paraffin; 
it will not grow on potato or carrot; it will 
not grow on acid-maltose agar nor on acid- 
glucose agar; attempts to grow it on nutrient 
agar and on BHI agar have given variable 
results; its optimum temperature is 37°C. It 
is delicate and is relatively slow growing; it 
is never hardy or richly branching, and it 
does not produce a surface scum or a con- 
fluent or filiform growth. It is a facultative 
anaerobe, differing in this respect from all 
other Nocardias described with the excep- 
tion of N. farcinica and N. rubropertincta. 

The author distinctive 
properties as follows: It produces a fairly 
compact colony composed of tangled myce- 
lium and exhibits radiating, clubbed, 
branched, and curled elements at the per- 
iphery. Fragmentation of the mycelium and 


summarizes its 


post-fission movement (zigzag arrangement ) 
occur at the periphery of the colony. Arthro- 
spores are produced. Stained preparations re- 
veal partial fragmentation into bacillary 
and coccoid forms. Mycelial forms and spores 
average slightly less than 1 mw in diameter. 
Neither nuclei nor septa were observed. 
Branching is of an alternate type. It is par- 
tially acid-fast. 

20. Nocardia flava (krassilnikov, 1988) 
Waksman and Henrici, 1948 (Krassilnikey, 


N. A. Bull. Acad. Sei. USSR No. 1: 139, 
1938). 
Not Proactinomyces flavus Humm_ and 


Shepard. 

Morphology: Cells at first filamentous, 
0.7 to 0.8 » in diameter; later, they break 
into long rods and then into cocei 0.7 w in 


THE GENUS 


diameter. Some strains form chlamydo- 
spores. Numerous inflated cells of the bulbi- 
form or fusiform type. Cell multiplication by 
fission, cross wall formation, rarely by bud- 
ding. Not acid-fast. 

Synthetic agar: Colonies bright yellow or 
golden. 

Nutrient agar: Growth dirty, lustrous, or 
rough and folded, of a dough-like consist- 
ency, yellow to straw-colored. No soluble 
pigment. 

Gelatin: No liquefaction. 

Milk: No coagulation and no peptoniza- 
tion. 

Starch hydrolysis: Shght. 

Sucrose: Weak inversion. 

Cellulose: No growth. 

Paraffin and wax: No growth. 

Fat: Weak growth, 

Habitat: Soil. 


21. Nocardia flavescens (Jensen, 1931) 
Waksman and Henrici, 1948 (Jensen, H. 
Proc. Linnean Soc. N. 8. Wales 56: 361, 
1931). 

Morphology: Substrate growth forms long, 
branched, nonseptate hyphae, 0.4 to 0.6. u. 
On nutrient agar and potato, septa are 
formed, mycelium fragmenting, partly re- 


sembling highly branched mycobacteria. 
Aerial mycelium consists of fairly long 


hyphae of the same thickness as the vegeta- 
tive hyphae, not very much branched, with- 
out spirals, often clinging together in wisps; 
hyphae break up into fragments of variable 
lengths, from 1.2 to 1.5 up to 10 to 13 ug, 
showing an irregular, granulated staining. 
Not acid-fast. 
Nutrient agar: 
and much wrinkled, first dirty cream-col- 


Substrate growth raised 


ored, later dark yellowish-gray, of a soft, 
moist, curd-like consistency. Aerial myce- 
lium absent. Soluble pigment absent. 
Glucose agar: Substrate growth super- 
ficial, wrinkled. honey-yellow, of a hard 
and cartilaginous consistency. Aerial myce- 


NOCARDIA 4: 


—~ 


lium thin, smooth, white. Soluble pigment 
yellow. 

Potato: Substrate growth 
and wrinkled, 


raised 

later 
yellowish-brown, soft and smeary. No aerial 
mycelium. No soluble pigment. 

Gelatin: Liquefaction slow. 

Milk: Coagulation; 
with acid reaction. 

Starch: Hydrolyzed. 

Cellulose: No growth. 

Paraffin: No growth. 

Sucrose: Inverted. 

Glucose broth: Rather secant growth. 
Granulated, yellowish sediment; no surface 
growth. Broth clear. No pigment. No acidity. 

Nitrate: Sight or no reduction. 

Source: Soil. 

22. Nocardia fordi (Erikson, 1935) Waks- 
man (Erikson, D. Med. Research Council 
Spec. Rept. Ser. 203: 15, 1935). 

Morphology: Substrate growth consists of 


much 


first cream-colored, 


slow peptonization 


filaments of medium length. Aerial myce- 
lium straight, sparse. Small 
spores on potato agar and starch agar. 

Glycerol nitrate agar: Growth thin, ex- 


short, oval 


tensive, golden brown, convoluted. 
Nutrient agar: small, 
to golden, ring-shaped; later, heaped-up 


Colonies creamy 
patches, becoming golden brown and con- 
voluted. 

Ege medium: Colonies minute, cream- 
colored, elevated, becoming golden brown, 
raised. 

Potato: Growth yellowish in thin terminal 
portion, tending to be piled up. Aerial myce- 
lium secant, white, at top of slant. Later, 
growth abundant, golden brown, confluent, 
partly honeycombed, partly piled up. 

Gelatin: No visible growth, slight soften- 
ing of gelatin; later partial liquefaction. 

Milk: Surface ring brownish. Coagulation 
positive. 

Starch: Not hydrolyzed. 

Source: Human spleen in a case of acho- 
luric jaundice. 


44 THE ACTINOMYCETES, Vol. II 


23. Nocardia formica Harris and Wood- 
ruff, 1953 (Harris, D. A. and Woodruff, H. 
B. Antibiotics Ann. 1953-1954, 609-614). 

Morphology: Mycehal development ex- 
tensive, with no fragmentation of hyphae. 
Ghost filaments and cytoplasmic condensa- 
tions produced. In submerged culture, 
straight and curved rods develop, exhibiting 
the Y- and V-forms. Rods are 0.9 to 1.1 by 
1.3 to 6.0 uw. Not acid-fast. 

Sucrose nitrate agar: Growth very faint 
or none at all. 

Glucose-asparagine Growth fair. 
Aerial mycelium grayish-white. Sporulation 
poor. 

Nutrient agar: Growth fair. Aerial myce- 
lium none. 

Peptone-glucose agar: Growth tannish- 
colored. Aerial mycelium white to grayish, 
gradually covering surface. Reverse side 
dark brown. Soluble pigment brown. 

Egg medium: Growth excellent, buff-col- 
ored, convoluted, moist. No liquefaction. 
Medium not discolored. 

Starch agar: Starch hydrolyzed. 

Gelatin: Liquefaction rapid. Soluble pig- 
ment none. Growth settled on bottom of the 
tube. 

Potato: Very poor growth. 

Nitrate reduction: Positive. 

Casein: Hydrolyzed. 

Paraffin: Not utilized. 

Optimum temperature: 28°C; good growth 
ats C: 


Carbon utilization: No acid production in 


agar: 


organic media from glucose, glycerol, lac- 
tose, maltose, and sucrose; acid produced in 
media trom glucose, glycerol, 


lactose, maltose, but not from sucrose. 


inorganic 


Antagonistic properties: Produces an anti- 
biotic substance active against Trichomonas 
and swine influenza virus. 

Source: Isolated from an abandoned nest 
of African ants in an imported mahogany 


log. 


24. Nocardia fructifera  (krassilnikov, 


1941) Waksman (Krassilnikov, N. A. Ac- 
tinomycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, 1941). 

Morphology: Growth not compact, mostly 
of dough-like consistency, smooth or rough. 
Hyphae breaking up into rods and in some 
cultures into cocci. Not acid-fast. Aerial 
mycelium well developed, whitish to rose- 
colored. Sporophores long, straight or weakly 
wavy, but not spiral-shaped. Spores cylin- 
drical, 1.5 by 0.7 jx. 

Synthetic agar: Growth rose-colored to 
bright red. No soluble pigment. 

Nutrient agar: Aerial mycelium weakly 
developed or absent entirely. 

Gelatin: Liquefaction slow. 

Milk: Coagulation positive; peptoniza- 
tion weak. 

Sucrose: Inverted. 

Starch: Hydrolysis weak. 

Cellulose: Poor growth. 

Paraffin: No growth. 

Fats: Good growth. 

Source: Soil. 

Remarks: One strain was obtained as a 
mutant of another Nocardia; another strain 
was changed, after 8 months of cultivation, 
into a typical Streptomyces. This species 1s 
considered as a transition form between the 
two genera. 


25. Nocardia gibsoni (Erikson, 1935) 
Waksman (Erikson, D. Med. Research Coun- 
cil Spec. Rept. Ser. 203: 36, 1935). 

Morphology: Young growing mycelium 
branches profusely at short intervals, finally 
grows out into long, frequently wavy fila- 
ments. Property of producing aerial myce- 
lium apparently lost. 

Nutrient agar: Colonies small, cream-col- 
ored, depressed, partly confluent, growing 
into an extensive wrinkled surface layer. 

Glucose nutrient agar: Growth cream- 
colored, wrinkled, membranous. 

Potato agar: Growth wrinkled, glistening, 
membranous. 

Blood agar: Colonies small, diserete, yel- 


THE GENUS 


lowish, irregularly wrinkled, clear hemolytic 
zone. 
small, 


medium: Colonies round, 


smooth, colorless, with conically elevated 


1 
Koo 
55 


centers. 

Potato: No growth. 

Gelatin: Dull white flakes sinking as me- 
dium liquefies. Liquefaction rapid. 

Milk: Coagulation positive; peptoniza- 
tion limited. 

Starch: Not hydrolyzed. 

Source: Human spleen in a case of acholuric 
jaundice. Injected into a monkey and reiso- 
lated. 

Type culture: ATCC 6852. 

26. Nocardia globerula (Gray, 1928) Waks- 
man and Henrici, 1948 (Gray, P. Proce. Roy. 
Soe. (London) B 102: 265, 1928). 

Morphology: Growth orange to orange- 
buff. It consists of curved rods and _fila- 
ments, | by 2 to9 uw, with many coccoid cells, 
especially in old cultures. Rods and filaments 
frequently irregularly swollen. Not acid-fast. 
Capsules may be present. 

Nutrient agar: Surface colonies irregu- 
larly round, 3 to 5 mm in diameter, convex, 
white, smooth, shining; edge undulate, erose. 
Deep colonies, lens-shaped. 

Gelatin: Surface colonies irregularly 
round, | to 2 mm in diameter, convex, light 
buff, smooth, shining. Stab: nailhead, irreg- 
ularly round, convex, pinkish-white, smooth, 
shining. 

Potato-glycerol agar: 
moist, smooth, pale pink. 

Milk: Alkaline. 

Nutrient and peptone broth: Turbid with 
viscous suspension. 

Nitrate reduction: None. 


Growth filiform, 


Ege medium: Growth spreading, raised, 
moist, orange-colored. 

Indole agar: Blue crystals of indigotin 
formed. 

Temperature: Optimum 25-28°C. 

Phenol: Utilized. 

Source: Soil. 


NOCARDIA 45 


Remarks: This organism resembles most 
closely N. corallina. It is distinguished by 
producing a more watery type of surface 
growth, more nearly entire deep colonies, 
and more particularly by the production of 
indigotin from indole. No acid from glucose, 
lactose, maltose, sucrose, or glycerol. 

Type culture: ATCC 13,130. 

27. Nocardia hortonensis (Erikson, 1935) 
Waksman (Erikson, D. Med. 
Council Spec. Rept. Ser. 203: 22, 1935). 

Morphology: Substrate growth made up 
of very slowly developing unicellular myce- 
lium, composed of long slender straight 
branching filaments. Aerial mycelium very 


Research 


sparse, forming straight hyphae only once 
on potato. Not acid-fast. 

Glycerol nitrate agar: Colonies coiled, 
colorless, lustrous patches, isolated, with 
central depression. 

Nutrient agar: Growth very slow, as few 
smooth, cream-colored, coiled colonies. 

Glucose nutrient agar: Growth as coiled 
and heaped up cream-colored translucent 
masses. 

Potato agar: Colonies colorless, blister; 
later dull green heaped and coiled mass. 
Medium becomes slightly discolored. 

Potato: Colonies abundant, 
umbilicated, round, some coiled in raised 


colorless, 


masses; later, liberal olive-green growth. 
Aerial mycelium dense, velvety gray-green 
at top of slant. 

Gelatin: Colonies round, cream-colored on 
surface and a few millimeters below. No 
liquefaction. 

Milk: Surface growth green; peptoniza- 
tion positive. Color at first purple, later 
brown. 

Source: From pus containing typical 
actinomycetic granules from parotid abscess. 

28. Nocardia intracellularis Cutting and 
MeCabe, 1949 (Cutting, J. T. and McCabe, 
A. B. Am. J. Pathol. 25: 1-47, 1949). 

Morphology: Filaments branched, becom- 


ing fragmented, composed of bacillary ele- 


AG THE ACTINOMYCETES, Vol. II 


ments in series, 0.2 to 0.45 w in width. Liquid 
cultures give branched colonies. The hyphae 
do not form club-shaped tips, and lack 
chlamydospores. Not discolored when 
stained with fuchsin and treated with acid 
alcohol. 

Agar media: Colonies 
wet-shining, smooth, and nonmucoid. 

Potato agar: No growth. 

Gelatin: Growth poor. No liquefaction. 

Glycerol broth: White, mucoid masses 
formed at bottom of tube. Medium remains 


circular, raised, 


clear. 

Milk: Acid after 20 to 30 days. 

Starch: Not changed. 

Tyrosinase reaction: Absent. 

Cellulose: Not decomposed. 

Nitrate reduction: None. 

Oxygen requirement: Does not develop 
in the absence of oxygen, but grows in an 
atmosphere having 10 per cent COs. 

Paraffin: Used as the only source of car- 
bon. 

Temperature: Grows at 37.5°C and _ tol- 
erates well temperatures up to 40°C. 

Habitat: Observed in granuloma of in- 
fected lymph nodes and in the feces of a 
living patient whose death it ultimately 
saused. Observed also at autopsy in widely 
disseminated granulomatous lesions which 
it produced. 

Type culture: ATCC 13,209. 


29. Nocardia ivorensis Combes, Kauff- 
mann and Vazart, 1957 (Combes, R., Kauff- 
mann, J., and Vazart, B. Compt. rend. 224: 
821-824, 1582-1587, 1957). 

Agar media: Substrate growth character- 
ized by the black coloration of its coccoid 
bodies, by their elongation, by their resist- 
ance to dryness and to heat, by their cellulo- 
lytic properties, and by their production of 
an orange pigment on different media. Col- 
omes at first whitish, centers becoming light 
orange to brown, later black. Black circles 
formed successively around the central circle 
and finally becoming confluent. Later, sur- 


face of colony is uniformly black, shiny, and 
waxy; at the periphery, grayish, radiant 
outgrowths develop in the agar, forming a 
more or less regular fringe; on the surface of 
this fringe brown, rapidly darkening, con- 
centric zones appear. 

Milk: Reddish-orange surface film, and 
isolated colonies adhering to the walls of 
the tube. 

Potato: Growth orange, 
places, with the appearance of coccoid forms. 

Gelatin: Growth scant, slow; later the 
culture is orange in color. Liquefaction 


darkening in 


positive. 

Cellulose (filter paper or washed cotton), 
moistened with the synthetic medium: Light 
brown colonies appear in 2 days and turn 
dark at 6 days, being entirely composed of 
coccoid forms; later, colonies are entirely 
black. Cellulose progressively disintegrates. 

Paraffin: White colonies appear at 5 days. 
They remain small and rapidly form coccoid 
elements. 

Nitrate reduction: Positive. 

Remarks: Three isolated cultures differed 
from each other mainly in the rapidity with 
which they formed coccoid elements. Or- 
ganism closely related to N. nzgra. 

Habitat: Colony of 
coast of Africa. 

30. Nocardia kurotshi Uesaka, 1952 (Ue- 
saka, I. J. Antibiotics (Japan) 5: 75-79, 
1952). 

Morphology: Aerial mycelium abundant. 
Sporophores slightly curved at first, later 


termites on ivory 


turning around each other. Acid-fast. 

Glycerol nitrate agar: Growth thin, pale 
yellow. Aerial mycelium punctiform, white. 
Soluble pigment yellow. 

Nutrient agar: Colonies wrinkled, grayish- 
yellow. No aerial mycelium. Soluble pigment 
faint grayish-brown. 

Glucose nutrient agar: Growth abundant, 
at first yellowish-brown, then reddish-brown. 
Aerial mycelium secant, white at margin of 


THE GENUS 


colonies. Soluble pigment red to wine- 
colored. 

Potato: Growth moderate, at first red or 
brownish red, later dark brown. Aerial myce- 


lium grayish white. Soluble pigment dark 


brown. 
Glucose broth: Red colonies forming 
pellicle. Abundant, flocculent sediment. 


Soluble pigment dark brown. 

Gelatin: Growth yellowish-brown, sinking 
into medium. No aerial mycelium. No lique- 
faction. Soluble pigment yellowish-brown. 

Milk: No coagulation. Slow peptonization. 
Brown pigment. 

Starch: Hydrolyzed. 

Carbon source: Lactose well utilized. 

Nitrate reduction: None. 

Antagonistic — properties: 
antibiotic neonocardin, active against vari- 


Produces an 


ous bacteria. 

Source: Soil. 

31. Nocardia letshmanii Chalmers and 
Christopherson, 1916 (Birt, C. and Leish- 
man, W. B. J. Hyg. 2: 120, 1902; Chalmers, 
A. and Christopherson, I. Ann. Trop. Med. 
Parasitol. 10: 255, 1916). 

Morphology: Initial cells frequently swol- 
len, large and irregular, aggregated in short 
chains and then branching out into regular 
narrow filaments; later entire colonies as- 
teroid in appearance, very fine and close 
angular branching, with aerial hyphae situ- 
ated singly. Whitish-pink aerial mycelium 
generally abundant with irregularly cylin- 
drical conidia. Acid-fast. 

Glucose nutrient agar: Colonies rounded, 
elevated, red, with paler frosting of sparse 
aerial mycelium. No soluble pigment. 

Glycerol agar: Colonies small, round, pink, 
tending to be umbilicated and piled up. 
Aerial spikes stiff, white. 

Potato agar: Colonies minute, colorless, 
round. Aerial mycelium white, in patches. 

Kgg medium: Growth colorless, confluent, 
with little wart-like 


studded projections 


NOCARDIA 47 


bearing stiff aerial spikes; later pinkish. 
Aerial mycelium white. Medium discolored. 

Gelatin: Colonies small, pink. No lique- 
faction. 

Milk: Surface growth; aerial mycelium 
white turning pink. Coagulum solid, later 
partly peptonized. 

Pathogenicity: To rabbits, rats, and 
guinea pigs. 

Source: Fatal case of lung disease and 
pericarditis in man. 

Remarks: According to Gonzalez Ochoa 
and Sandoval (1956), N. 
synonym of .V. asteroides. 


32. Nocardia listeri (Erikson, 1935) Waks- 
mann (Erikson, D. Med. Research Council 
Spec. Rept. Ser. 203: 23-24, 1935). 

Morphology: 
straight. Spores oval. 

Glycerol nitrate agar: Growth abundant, 


leishmanii is a 


Sporophores short and 


moist, cream-colored. Aerial mycelium pow- 
dery, white, with exuded drops. 
Calcium malate agar: Growth poor, in 
form of a biscuit-colored membrane. 
Nutrient Growth 
cream-colored, 


agar: smooth, moist, 


margin depressed, center 
elevated. 

Glucose nutrient agar: Growth cream- 
colored, glistening. 

Potato agar: Growth extensive, colorless, 
warted surface. Dirty pink coloration after 
2 weeks. Scant white aerial mycelium after 
4 months. 

Potato: Growth abundant, dull, brownish, 
wrinkled. Aerial mycelium white. 

Gelatin: Surface colonies round, white; 
after 45 days, confluent skin. Liquefaction 
slight. 

Blood agar: Colonies small, round, cream- 
colored, with smooth, translucent surface. 
No hemolysis. 

Serum agar: Colonies small, irregular, 
moist, cream-colored, tending to be heaped 
up; later somewhat transparent. 

Milk: Coagulated. No change in reaction. 


Source: From human material. 


48 THE ACTINOMYCETES, Vol. II 


33. Nocardia lutea Christopherson and 
Archibald, 1918 (Christopherson, J. B. and 
Archibald, R. G. Lancet 2: 847, 1918).* 


Morphology: Growth consists of irregular, 
spreading, polymorphous colonies, compris- 
ing swollen and segmented cells of all shapes 
and sizes with markedly granular contents. 
Later cells more monomorphous, the fila- 
ments being arranged in angular apposition. 

Glycerol nitrate agar: Growth in form of 
yellowish-pink, wrinkled membrane. 

Nutrient agar: Growth abundant, co- 
herent, moist, pink, membranous with round 
discrete colonies at margin. 

Glucose nutrient agar: 
reddish, smeary. 

Potato agar: Small filamentous colonies 
are formed; irregular angular branching. 
Aerial hyphae few, isolated, short, straight. 

Potato: Growth carrot-red, moist, thick, 
granular in bands, partly raised and with 
discrete round colomes. Aerial mycelium 
sparse, colorless, very thin at top of slant. 

Gelatin: Growth pale pink, wrinkled on 
wall of tube. Colorless punctiform and stel- 
late colonies in medium. No liquefaction. 

Milk: Growth orange-red on surface and 
at bottom. 


Growth secant, 


Ege medium: Growth poor, dull pink. 

Source: Actinomycosis of the lachrymal 
gland. 

Remarks: According to Erikson, various 
saprophytes, such as N. rubra and N. poly- 
chromogenes, are closely related. 


34. Nocardia marina (Krassilnikov, 1949) 
Waksman (Humm, H. J. and Shepard, K. 
S. Duke Univ. Marine Sta. Bull. 3: 76, 
1946; Ixrassilnikov, N. A., Guide to the 
identification of bacteria and actinomycetes, 
Moscow, 1949). 

Synonyms: Proactinomyces flavus Humm 
and Shepard. Proactinomyces citreus marinae 
Krassilnikov. 

* Description after Erikson, D., Med. Res. 
Council Spee. Rept. Ser., 203: 30, 1935. 


Morphology: Growth smooth, — bright 
yellow color, of a dough-like consistency. 
Hyphae long, filiform, branching, breaking 
down into short rods and cocci. No aerial 
mycelium. 

Synthetic and protein salt water media: 
Good growth. 

Gelatin liquefaction: Positive. 

Agar: Liquefied. 

Milk: No coagulation; peptonization posi- 
tive. 

Nitrate reduction: None. 

Starch: Hydrolyzed. 

Carbon sources: Acetic, lactic, and butyric 


acids utilized. Acid formed from various 
sugars. 


Temperature: 25-30°C. 
Habitat: Atlantic Ocean marine deposit. 


35. Nocardia mesenterica (Orla-Jensen, 
1919) Waksman and Henrici, 1948 (Orla- 
Jensen, S. The lactic acid bacteria, 1919, 
181; Jensen, H. L. Proc. Linnean Soc. N.S. 
Wales 57: 373, 1932). 

Morphology: Growth forms extensive my- 
celium composed of richly branching hyphae 
of a somewhat variable thickness, 0.4 to 0.8 
u. No aerial mycelium. Later, hyphae divide 
into fragments of varying sizes and shapes, 
partly diphtheroid rods, but no real cocci. 
There is, particularly in complex organic 
media, a tendency to form large, swollen, 
fusiform to almost spherical cells, up to 3.5 
uw in diameter. These may stain intensely 
with carbol fuchsin. 

Glucose-asparagine agar: Growth fair, 
raised, granular, very pale yellow, glistening. 
Condensation water-clear. 

Glucose-peptone agar: Growth excellent, 
spreading. At first flat and smooth, pale 
straw-yellow, perfectly hard and cartilagin- 
ous, later raised and strongly folded, of a 
loose, curd-like consistency, bright lemon- 
vellow. 

Potato: Growth 
cream-colored smear. 

Gelatin: Growth 


seant, restricted, soft, 


finely arborescent, 


THE GENUS NOCARDIA 49 


cream-colored in the stab. Surface colony 
raised, folded, pale yellow. No liquefaction. 

Milk: Small cream-colored granules along 
the tube. No proteolytic action. 

Starch: Hydrolyzed. 

Cellulose: Not utilized. 

Nutrient broth: Good growth; volumi- 
nous, flaky, whitish sediment; broth clear. 

Nitrate reduction: Negative. 

Sucrose: Inverted. 

Source: Fermented beets. 

Remarks: Sodium _ nitrate, 
phosphate, and asparagine are utilized, al- 
though these are inferior to peptone as 


ammonium 


sources of nitrogen. 


36. Nocardia muris (Schottmiiller, 1914) 
de Mello and Pais, 1918 (de Mello and Pais. 
Arq. Hig. Pat. Exot. 6: 183, 1918). 

Synonyms: Streptothrix muris-ratti Schott- 
miller, 1914. Streptobacillus moniliformis 
Levaditi, 1925. Actinomyces muris Topley 
and Wilson, 1946. 
Krassilnikov, 1941. 

Morphology: Slender branching filaments, 
0.4 to 0.6 uw in diameter, breaking up into 
rods and cocci. Often cells form long chains 
of bead-like cells, with terminal club-like 


Proactinomyces  muris 


swellings. Nonacid-fast. 

Growth: None on ordinary media. Growth 
occurs in presence of serum, ascitic fluid or 
blood. 

‘Nutrient agar: No growth. 

Glucose agar: No growth. 

Serum agar: Grayish-yellow, clear col- 
onies, 0.2 to 0.3 mm in diameter, with 
smooth, glistening surface and entire edge. 
Yasily emulsifiable. 

Gelatin: No growth. 

Potato: No growth. 

Milk: No effect. 

Nitrate reduction: None. 

Blood agar: Like serum agar. No he- 
molysis. 

Egg medium: Similar to growth on serum 
agar. No liquefaction. 


Oxygen: Grows aerobically. Grows also 
under anaerobic conditions. 

Acid production: Acid produced in serum 
agar media, with glucose and salicin, some- 
times with maltose and lactose. 

Habitat: Parasite inhabiting nasopharynx 
of rats. Isolated from body of patient bitten 
by a rat. 

temarks: Similar organisms by a variety 
of names, such as A. putoriz, were also 
listed. Above description based on data of 
Topley and Wilson (1946). 

37. Nocardia nigra (Krassilnikov, 1941) 
Waksman (not N. 
Chalmers; not Streptothrix nigra 
Doria.) (Krassilnikov, N. A. Actinomy- 
cetales. Izvest. Akad. Nauk. SSSR, Moskau, 
1941). 

Morphology: Growth rough, folded, shiny, 
dough-like consistency. Cells thread-like, 
breaking up readily into rods 2 to 10 by 
0.7 w and cocci, 0.6 to 0.8 p. No aerial 
mycelium. Gram-positive, not acid-fast. 


nigra Castellani and 


LOssl- 


Agar media: Growth poor, at first color- 
less or brownish, gradually becoming darker, 
later dark brown and even black. Pigment 
not excreted in medium. Many cells are 
swollen to 3 uw in diameter. 

Potato: Growth good. 

Gelatin: No growth. No liquefaction. 

Milk: No change. 

Cellulose: No growth. 

Paraffin and wax: No growth. 

Nutrient broth: Small sediment produced. 
Medium clear. 

Carbon utilization: Utilizes glucose and 
mannose, with formation of acid. 

Source: Seldom found in soil. 

Remarks: Culture rapidly loses its  vi- 
ability on continued cultivation. 


38. Nocardia opaca (den Dooren de Jong, 


1927) Waksman and Henrici, 1948 (den 
Dooren de Jong, L. E. Centr. Bakteriol. 


Parasitenk. Abt. II, 71: 216, 1927: Jensen, 
H. L. Proc. Linnean Soc. N.S. Wales 57: 
369, 1932). 


oO THE ACTINOMYCETES, Vol. II 


FIGURE 13. 


N. opaca (N. erythropolis), showing scheme of branching; 


glycerol nutrient agar, first 


sketch 10 hours incubation; others at hourly intervals (Reproduced from: McClung, N. M. Lloydia 12: 


153, 1949). 


Svnonyms: Nocardia crystallophaga (Gray 
and Thornton); N. 


Thornton); Preactinomyces opacus (Jensen). 


erythropolis (Gray and 


Morphology: Growth lustrous, rose-col- 
ored to red. Hyphae long, curved, irregular 
and branching, breaking up into rods and 
cocci. Not acid-fast. Gram-positive (lig. 13). 

Potato-glycerol agar: Growth dry, rough, 
crumpled, pink to buff-colored. 

Gelatin: Colonies round, convex, whitish, 
smooth, shining, with edges shghtly ar- 
borescent. Stab: convex, whitish, smooth, 
resinous, filiform, erose. No liquefaction. 

ge medium: Growth spreading, smooth, 


moist, salmon-colored. 


Potato: Growth covered with tufts. of 
aerial hyphae. 

Milk: Grayish pellicle. No coagulation, no 
peptonization. Reaction slightly alkaline. 

Nitrate reduction: Positive. 

Starch: Not hydrolyzed. 

Sucrose inversion: Negative. 

Carbon sources: Saturated, long chain 
aliphatic hydrocarbons are utilized as sources 
of energy. 

Temperature: Optimum 30°C. 

Source: Seldom found in soils. 

Differs from WN. 


N. polychromogenes in that the 


Remarks: corallina and 
cells are 


much longer than those of the former and 


THE GENU 


much shorter than those of the latter. Erik- 
son (1949) added the following character- 
istics: Soft cream to pink growth on nutri- 
ent agar media. On synthetic media, growth 
colorless and thin, producing an initial myce- 
lium, the hyphae dividing rapidly into short 
rods; addition of 0.01 per cent MnSQO, stim- 
ulated production of pale pink pigment. 
Acid-fast cell elements predominated during 
periods of maximum growth and free air 
supply. A study of the morphology of NV. 
opaca grown on hydrocarbons and _ fatty 
acids has been made by Webley (1955). 


39. Nocardia panjae (Erikson) Waksman 
(Erikson, D. Med. Research Council Spec. 
Rept. Ser. 203: 1935, 16-17). 

Morphology: Substrate growth made up 
of very small, round colonies; unicellular 
mycelium with slender, branching filaments. 
Aerial mycelium not visible on any medium, 
but occasional isolated aerial branches. 

Glycerol nitrate agar: Growth poor; scant 
colorless patch. 

Calcium malate agar: Growth colorless to 
pink, spreading; later, bright red mass, 
buekled and shining, colorless submerged 
margin. 

Nutrient agar: Growth irregularly piled 
up, convoluted, colorless, easily detachable, 
brownish. 

Glucose agar: Small colorless coiled mass, 
later heaped up as green growth. 

Gelatin: Liquefaction rapid. 

Milk: Surface growth pale green. Coagula- 
tion and peptonization. 

Potato agar: Growth as small elevated, 
convoluted, colorless mass with purple tinge 
in center. 


Egg medium: Colonies small, round, 
tough, colorless; margin well embedded. 


Later, colonies elevated, warted, darkened, 
medium discolored and broken. Slight de- 
gree of liquefaction; medium dark brown. 


Source: An uleer of the abdominal wall of 


a patient in India. 


) 


NOCARDIA 51 
1951) 
H. 


Wales 56: 362, 


(Jensen, 
1948 (Jensen, 
Nexo: 


40. Nocardia para ffinae 
Waksman and Henrici, 
Proce. Linnean Soc. 
1931). 

Morphology: Growth hard, firm, yellow- 
ish, consisting initially of an extensive myce- 
lium, with long, richly branching hyphae, 
0.4 to 0.5 uw thick. After 5 to 6 days, numer- 
ous end double 
thickness, and divide into oval, spore-like 
elements, 0.8 to 1.0 by 1.2 to 1.5 uw. Division 


branches swell to about 


starts at the tips of the swollen branches 
and proceeds basipetally until most of the 
hyphae appear divided. Primary septa have 
not been seen in the hyphae. The spore-like 
elements are markedly acid-fast. Aerial my- 
celium white consisting of short, straight, 
not very much branched hyphae, 0.4 to 0.6 
u thick, which never show any differentiation 
into spores (see also Erikson, 1949). 
Sucrose nitrate agar: Growth very scant, 
as thin colorless veil. Aerial mycelium trace, 
white. 
fair, 
flat, growing into medium; pale ocher-yellow 


Glucose-asparagine agar: Growth 
to orange, with raised outgrowths on the 
surface. Aerial mycelium secant, white. 

Nutrient agar: Growth slow, somewhat 
raised, ocher-yellow, hard, smeary surface 
loose. Aerial mycelium scant, small white 
tufts. No soluble pigment. 

Potato: Growth mycelium granulated, 
first pale yellow, later deep ocher-yellow to 
orange. Aerial mycelium scant, white. No 
soluble pigment. 

Gelatin: No liquefaction. 

Milk: No coagulation; no peptonization. 

Starch: No hydrolysis. 

Cellulose: Not decomposed. 

Paraffin: Readily utilized. 

Nitrate reduction: Negative. 

Sucrose: Not inverted. 

Liquid media (milk, broth, synthetic solu- 
Small, 


yellow to orange colors, firm but can be 


tions): round granules of various 


crushed into a homogeneous smear. In old 


o2 THE ACTINOMYCETES, Vol. II 


Ficure 14. Orskov’s motile Nocardia (Reproduced by courtesy of N. M. McClung). 


broth cultures, a thick, hard, 
brownish surface pellicle is formed. 


Habitat: Soil. 
41. Nocardia petroleophila Hirsch and 
Engel, 1956 (Hirsch, P. and Engel, H. Ber. 
deut. bot. Gesell. 69: 441-454, 1956). 
Morphology: Grows slowly, but 


orange to 


abun- 
dantly on all mineral media, faster in a 
petroleum atmosphere. It grows on certain 
organic media, but does not produce any 
aerial mycelium. Substrate mycelium breaks 
up readily into rods; ‘‘involution cells” are 
formed abundantly. Mycelial threads are 
long, monopodially branched, 0.6 to 1.2 u 
in diameter, and contain ‘‘metacleromatic 
granules,” readily stained with aqueous 
methylene blue. Aerial hyphae have the 
same diameter as the substrate hyphae, 
little branched. Aerial mycelium wets with 
difficulty. No aerial spores. Mycehal seg- 
ments 1.2 to 5.0 by 1.2 to 12.5 uw. Gram-posi- 
tive; not acid-fast. 

Glucose-asparagine agar: Growth limited. 
Colonies 0.3 mm, white, yellow reverse. 
Aerial mycelium snow-white. Soluble pig- 


ment none. 


Caleium malate agar: Growth limited. 

Nutrient agar: Growth limited. Colonies 
whitish-yellow, 0.5 mm. No aerial mycelium. 

Starch agar: Growth limited. Aerial my- 
celum snow-white; reverse yellowish. Starch 
not hydrolyzed. 

Potato: No growth. 

Gelatin: Growth in form of microscopic 
colonies. No liquefaction. No pigmentation. 

Milk: 


No aerial mycelium. No coagulation; no pep- 


Growth limited, yellowish, dry. 


tonization. 


Temperature: Optimum 25-28°C. Re- 
sistant to drying. 
Salt concentration: Resistant to high 


concentration. 
Habitat: Soil. 


42. Nocardia polychromogenes (Vallée, 
1903) Waksman and Henrici, 1948 (Vallée, 
H. Ann. inst. Pasteur 17: 288-292, 1903; 
Jensen, H. Proc. Linnean Soc. N.S. Wales 
56: 79, 363, 1931). 

Morphology: Growth bright red, coral- 
red to red-pink, of a doughy consistency, 
later becoming leathery. Aerial mycelium 
whitish with pink hue. Substrate growth 


THE GENUS 


forms long wavy filaments, 0.4 to 0.5 by 
70 to 100 uw, extensively branched but with- 
out septa. Older cultures consist entirely of 
rods, 4 to 10 uw, frequently in V-, Y-, or 
smaller coceoid forms. Gram-positive, not 
acid-fast, frequently showing bands and 
granules. 

Nutrient agar: Growth scant, orange-red. 
No aerial mycelium. No soluble pigment. 

Glucose agar: Growth raised, flat, glisten- 
ing, rose-colored, later becoming folded and 
coral-red. 

Gelatin: Growth along stab thin, yellow- 
ish, with thin radiating filaments. Surface 
growth flat, wrinkled, red. No liquefaction. 

Milk: Growth 
colored surface granules, later forming a 
thick, soft, orange-colored sediment. No 
coagulation; no peptonization. 

Starch: Hydrolyzed. 

Paraffin: Utilized. 

Cellulose: No growth. 

Temperature: Optimum 22-25°C. 

Source: Blood of a horse; soil in France 
and Australia. 

Remarks: Differs from NV. corallina in the 
formation of very long filaments and in fili- 


starts as small orange- 


form growth in gelatin stabs. 
Type culture: IMRU 3409. 


43. Nocardia pulmonalis (Burnett, 1909) 
Waksman and Henrici, 1948 (Burnett, 
S. H. Ann. Rept. N. Y. State Vet. Coll. 
1909-1910, 167). 

Morphology: Mycelium acid-fast, espe- 


cially in early stages of growth; breaks up 


readily into oval-shaped cells. Growth 
lemon-yellow with white aerial mycelium. 
Consistency of colonies leathery. 

Peptone-beef extract agar: Growth moist, 
raised, in form of small, spherical colonies. 

Glucose-peptone-beet extract agar: 
Growth dull, whitish, convoluted. 

Potato: Growth abundant, in form. of 
small, translucent, round colonies, becoming 


lemon-yellow. Later, growth becomes con- 


NOCARDIA de 


we 


voluted or folded with chalky white aerial 
mycelium. Color of plug brownish. 

Gelatin: Colomes small, whitish, spheri- 
cal; edges of colony becoming chalky white. 
Limited liquefaction. 

Milk: Colonies on surface of the medium. 
Coagulation and gradual peptonization. 

Pathogenicity: Nonpathogenic for rabbits 
and guinea pigs. 

Source: Lungs of a cow. 


44. Nocardia rangoonensis (Erikson, 1935) 
Waksman and Henrici, 1948 (Erikson, D. 
Med. Research Council Spec. 
203: 33-34, 1935). 

Morphology: Growth consists of branch- 
ing hyphae which segment and _ present 


tept. Ser. 


sipping and angular arrangement. Aerial 
hyphae few, short, straight, later developing 
into a profusely branching, long, waving 
aerial mycelium. Not acid-fast. 

Glycerol nitrate agar: Growth dull, mealy, 
pink, wrinkled. Aerial mycelium 
white. Medium slightly discolored. 

Nutrient Colonies round, 
umbilicated, raised, cream-colored to pale 


secant, 


agar: lobate, 
pink. Later, colonies colorless, medium dis- 
colored dark brown. 
nutrient 
voluted, coherent, cream-colored; medium 
discolored. Later, growth wrinkled, biscuit- 
colored, colorless margin. Aerial mycelium 
on border, white. Soluble pigment dark 


Glucose agar: Growth  con- 


brown. 

Potato agar: Colonies small, round, lemon- 
colored, partly confluent. Submerged growth 
greenish. Aerial mycelium white. Medium 
colored light brown. 

Egg medium: Growth extensive, colorless. 
Aerial mycelium in center, pale pink. 

Gelatin: Colonies abundant, minute in 
medium; larger, cream-colored colonies on 
surface. Aerial mycelium white. Brown pig- 
ment surrounding growth. No liquefaction. 

Milk: Surface ring yellow. Coagulation 
positive; peptonization partial. Soluble pig- 
ment dark brown. 


o4 THE ACTINOMYCETES, Vol. II 


Source: Human pulmonary case of strepto- 
thricosis. 


45. Nocardia rhodnii (Erikson, 1985) 
Waksman and Henrici, 1948 (Erikson, D. 
Med. Research Council Spec. Rept. Ser. 
203: 29, 1935). 

Morphology: Substrate growth made up 
of minute colonies, composed of hyphal seg- 
ments arranged in angular apposition. Aerial 
mycelium is short and straight. Later, 
growth becomes extensive and spreading, 
made up partly of long, branching filaments, 
and partly of short segments exhibiting 
branching, each giving rise to 
aerial hyphae. Angular branching very 
marked, delicate, spreading, herringbone 
patterns being formed. 

Sucrose nitrate agar: Colonies minute, 


shipping 


colorless, round. 
Glucose-asparagine agar: Growth abun- 
dant, coral-pink, convoluted, piled up. 
Glycerol agar: Growth made up of dull 
pink colonies, round and umbilicated, be- 
coming piled up and deeper coral-red. 
Potato agar: Growth abundant, 
piled up and stiff. Aerial mycelium white at 


pink, 


top of slant. 


medium: Membrane salmon-pink, 


Kee 
455 


granular, 

Gelatin: Colonies pale pink, in form of 
surface pellicle and as sediment. Liquefac- 
tion rapid. 


Milk: Growth bright orange. Medium 
unchanged. 
Nutrient broth: Salmon-pink flakes im 


sediment and colonies on surface. Medium 
discolored. 

Source: From reduviud bug, Rhodnius 
prolixus. 


Type culture: IMRU 653. 


46. Nocardia rubra (Actinomyces ruber 
sterilis Krassilnikov, 1949) Waksman (Kras- 
siinikov, N. A. Guide to the identification of 
bacteria and actinomycetes. Moskau, 1949). 


Agar media: Growth red, smooth, nodular, 


slightly lustrous with a gravel-like appear- 
ance. No aerial mycelium produced under 
laboratory conditions. Most strains form no 
soluble pigment. Some produce a brownish 
substance. Slightly acid-fast (Fig. 12). 

Milk: Unchanged. 

Krassilnikov examined 25 different strains 
said to belong to this type, but differing 
from one another in intensity of color and in 
certain physiological properties. He believed 
that under certain conditions of growth these 
cultures would develop an aerial mycelium 
and proper sporulation. 

This group was divided by Krassilnikov 
into four subgroups: 

a. Flat, compact colonies, red to pink in 
color, pigment insoluble. Gelatin not lique- 
fied, milk unchanged or only peptonized; 
starch not decomposed; nitrate not reduced. 

b. Colonies raised, dry, crumbling at con- 
tact with loop; red to brownish-red in color. 
Ready growth on synthetic media. Gelatin 
liquefied slowly; milk coagulated shghtly or 
only peptonized; nitrate reduced to nitrite; 
ready growth in paraffin and fats. 

c. Colonies compact, growing deep into 
substrate; pink to ight red in color; brown 
substance excreted into substrate. Gelatin 
shghtly liquefied; milk peptonized by some 
strains, starch slightly decomposed; nitrates 
not reduced to nitrites. Some cultures grow 
slowly in cellulose. Do not grow on paraffin. 

d. Flat or nodular colonies, growing com- 
pactly into medium. Frequently develop 
coremia on the surface; these consist of 
thickly interwoven sterile hyphae. The cul- 
tures grow poorly on artificial media. Gelatin 
liquefied or only milk not 
changed or only shghtly peptonized; starch 


not slowly; 
not decomposed. No growth on cellulose. 
Ready growth on fats, paraffin, and wax. 
temarks:  IXrassilnikov this 
eroup to comprise transition forms between 


considers 
Streptomyces and Nocardia. N. corallina 1s 
believed to be a related form; a number of 


synonyms are listed such as NV. agrestis, N. 


THE GENUS 


minima, and others. According to Schneidau 
and Shaffer (1957), this form does not pro- 


duce urease, whereas .V. corallina does. 


47. Nocardia rubropertincta  (Hefferan, 
1904) Waksman and Henrici, 1948 (Grass- 
berger, R. Miinch. med. Wochschr. 46: 343, 
1899: Hefferan, M. Centr. Bakteriol. Parasi- 
tenk. Abt. II, 11: 460, 1904). 

Morphology: Growth in form of small rods 
0.3 to 0.9 by 1.5 to 3.0 uw, showing angular 
arrangement; later, nearly coccoid, 0.6 by 
0.8 u. Tendency for branching on glycerol 
agar, but branching does not occur com- 
monly, though granules of aerial mycelium 
are sometimes seen. Not acid-fast or varia- 
ble. 

Nutrient agar: Colonies small, granular, 
becoming pink to red, depending on com- 
position of agar. 

Potato: Growth slow but excellent, inten- 
sive red, becoming dull orange. 

Gelatin: Colonies irregular with crenate 
margin and folded surface, coral-red. Growth 
in stab at first thin, then granular to arbores- 
cent with chromogenesis. No liquefaction. 

Milk: Surface seales thick, fragile, dull 
coral-red ; Milk 
somewhat viscid after 3 to 4 weeks. 

Nutrient broth: Faint uniform turbidity 
with salmon-pink pellicle, which is renewed 
on surface as it settles to form a red sedi- 


also sediment. becomes 


ment. 

Nitrate reduction: None. 

Carbon sources: Utilizes benzene, petro- 
leum, paraffin oil, and paraffin. 

Temperature: Grows well between 20 and 
Bw. 

Oxygen requirement: Aerobic to faculta- 
tively anaerobic. 

Source: Isolated from butter, soil, and 
contaminants of tuberculin flasks. 

femarks:  Mycobacterium-like —colomes 
with coral to vermillion-red chromogenesis 
on various media. 


48. Nocardia rugosa DiMarco and Spalla, 


. 
~ 


NOCARDIA 
1959 (DiMarco, A. and Spalla, C. Lab. 
Ricerche Farmitalia, Milano, 1959). 

Morphology: Hyphae short, 0.6 to 0.8 u 
in diameter, wavy, later angular, radiating 
from a center. After 20 to 24 hours, they 
break up into rods 8 to 20 uw long. No 
aerial mycelium. Nonacid-fast. 

Glucose-asparagine agar: Growth color- 
less, raised, moist, wrinkled. 

Glycerol Abundant, — lichenoid 
growth, dull cream. No consistency. Red- 
dish-brown soluble pigment after 15 days. 

Nutrient agar: Thick, cream-colored pel- 
licle, rough and folded. Dough-like con- 


agar: 


sistency. After 15 days, brown soluble pig- 
ment. 

Potato agar: Growth smooth, folded, with 
wrinkled and lichenoid portions. Colorless. 
Soft consistency. 

Milk: Coagulation; no peptonization. 

Gelatin: Liquefaction positive. 

Nitrate reduction: Negative. 

Starch: Nondiastatic. 

Sugar utilization: See Table 4. 

Optimum temperature: 84°C. 

femarks: Nonpathogenic. Produces vita- 
min By». 

Habitat: Cattle rumen. 

Type culture: IMRU 3760. 

This species was described further by 
Spalla (1959) as follows: It produces a color- 
less growth on glycerol, glucose, asparagine, 
and N-Z-amine agars. Acid is produced from 
qe 


arabinose, glycerol, d-mannitol, and adoni- 


glucose, galactose, ribose, rhamnose, 
tol; but not from d-mannose, sucrose, malt- 
inulin, 
Ni- 
trate is not reduced. Starch is not hydro- 


ose, lactose, trehalose, rafhinose, 


d-sorbitol, inositol, dulcitol, or salicin. 
lyzed. Gelatin is liquefied. Milk is coagu- 
lated. The organism will resist a temperature 
of 60°C for 115 hours, but not 3 hours. It is 
gram-positive. The terminal fragments are 
1.56 + 0.266 u. No aerial mycelium is pro- 
duced. 


49. Nocardia salmonicolor (den Dooren de 


18) THE ACTINOMYCETES, Vol. II 


Ficure 15. N. 


salmonicolor, growing on a hanging microdrop of liquid paraffin (a) surrounded by 


sucrose nitrate salt solution; (b) same plus 1.5 per cent agar (Reproduced from: Webley, D. M. J. Gen. 


Microbiol. 8: 71, 1953). 


Jong, 1927) Waksman and Henrici, 1948 
(den Dooren de Jong. Centr. Bakteriol. 
Parasitenk. Abt. I, 7h: 216, 1927): 
Morphology: Growth made up of short 
mycelium disintegrating into rods and cocci. 
Aerial mycelium sometimes stretching into 
quite long filaments, with small refractive 
granules. Many cells at the edge of the 
colonies show club- or pear-shaped swellings, 
up to 2.5 to 3.0 uw in width; many of these 
swollen cells later germinate with the forma- 
Acid- 


fastness is found among the earlier stages of 


tion of two more. slender sprouts. 
growth, especially in some of the strains and 
on some media (Fig 15). 

Glucose-asparagine agar: Growth — re- 
stricted, rather flat, edges lobate, surface 
warty, glistening; at first pale orange, later 
ocher-yellow; consistency crumbly. 

Glucose-peptone-beet extract agar: 
Growth excellent, of a doughy consistency, 
spreading, flat, dense, edges lobate, surface 
folded, glistening, yellow, gradually chang- 
ing to salmon-pink and deep orange-red. 

Potato: Growth good, raised, warty, 
crumbly, glistening, at first buff, changing 


to orange, and finally to almost blood-red. 


Gelatin: Growth in stab secant, arbores- 


cent. Surface colonies small, wrinkled, 
orange. No liquefaction. 
Milk: Pellicle of small cream-colored 


granules, later a thick orange sediment. No 
coagulation and no peptonization, although 
milk appears slightly cleared, the reaction 
becoming alkaline. 

Starch: Not hydrolyzed. 

Sucrose: Not inverted, although readily 
utilized with sodium nitrate as a source of 
nitrogen. 

Paraffin: Readily utilized as a source of 
carbon. 

Cellulose: No growth. 

Phenol: Not utilized. 

temarks: A detailed study of the acid- 
fast properties of this species has been made 
by Erikson (1949). It closely resembles V. 
corallina. 


sebivorans Erikson, 1954 
Pathol. Bacteriol. 68: 387- 


50. Nocardia 
(Erikson, 1D. J. 
393, 1954). 

Morphology : partially 
Aerial mycelium white, with 
characteristic nonwetting properties. Both 


Gram-positive, 
acid-fast. 


substrate and aerial mycelium show spon- 


THE GENUS 


taneous segmentation into shorter and 
longer cells of coccoid or bacillary dimen- 
sions. 

Agar media: Colonies firmly attached to 
medium. No soluble pigment. No acid pro- 
duced from glucose, mannitol, lactose, 
sucrose, starch, raffinose, galactose, rham- 
nose, sorbitol, maltose, dulcitol, glycogen, or 
glycerol. 

Sucrose nitrate agar: Growth fair; nitrate 
utilized. 

Gelatin: No liquefaction. 

Carbon utilization: Paraffin is good source 
of energy, also n-dodecane. Cresols not 
utilized. 

Temperature: Can withstand exposure to 
90°C for 10 minutes in a phosphate buffer 
suspension; denser suspensions withstood 3 
minutes at 100°C (Erikson, 1955). 

Habitat: Pus in a case of empyema. 

Pathogenicity: Rabbits and guinea pigs, 
slightly for mice. 

Remarks: Cells have avidity for oily sub- 
stances (lipophilic). Under unfavorable con- 
ditions of growth, on the surface of solid 
paraffin, large clubs and hexagonal cells are 
produced (Erikson, 1955). 

Type culture: NCTC 8595. 

51. Nocardia serophila (Sartory — and 
Bailly, 1947) emend. Waksman (Sartory, A. 
and Bailly, C. Compt. rend. 224: 1533-1534, 
1947). 

Morphology: Hyphae 
growth, much branched, occasionally curved, 
0.4 to 0.5 uw in diameter. Nonmotile. Rarely 
certain secondary branches are spiral shaped. 
intracellular arthrospores. 


produce angular 


Terminal and 
Gram-positive. Acid and alcohol resistant. 

Growth characteristics: Grows with diffi- 
culty on ordinary solid or liquid media; 
grows well on serum or blood media. 

Liquid peptone and serum media: Non- 
viscous, cream-colored growth, detaches 
from tube by agitation, medium remaining 
clear. 

Coagulated serum media: Growth in form 


NOCARDIA 57 


of small colonies, white at first, later be- 
coming yellowish cream-colored. 

Biochemical properties: Indole negative, 
HS positive, neutral red not reduced. Sugars 
like glucose, sucrose, lactose, galactose, and 
mannose not attached. 

Milk: Coagulation positive; peptonization 
positive. 

Coagulated serum: Not liquefied. 

Nitrate reduction: Positive. 

Oxygen demand: Strict aerobe. 

Optimum temperature: 35-37°C. 

Pathogenicity: Pathogenic to guinea pigs 
and rabbits. 

Habitat: Isolated from urine of patient 
suspected of renal tuberculosis. 


52. Nocardia sumatrae (Snijders emend. 
Erikson) (N. cuniculi, Snijders, Geneesk. 
Tijdsch. Ned. Indie 64: 47, 75, 1924). 

Not Streptothrix cuniculi Schmorl, 1891; 
not Nocardia cuniculi de Mello, 1919. 

Morphology: Growth made up of large 
swollen cells, giving rise to ramifying fila- 
ments or to small chains of short, thick seg- 
ments which branch out into more regular 
hyphae. Sometimes the irregular elements 
are beset with spiny processes before giving 
rise to typical long, branching filaments. 
Later the picture becomes more mono- 
morphous, and short straight aerial hyphae 
are borne, which presently exhibit irregular 
segmentation. 


Glycerol nitrate agar: Colonies small, 
round, elevated, cream-colored, margins 


depressed; becoming smooth, discrete, yel- 
lowish. 

Glucose-asparagine agar: Colonies minute, 
colorless, becoming dull pink, partly con- 
fluent and piled up. Aerial spikes few, stiff, 
pink 


Nutrient agar: Colonies small, round, 
elevated, cream-colored, umbilicated and 
radially wrinkled. 

‘gg medium: Growth scant, pinkish, 


smeary. 
Potato: Growth coral-pink, dry, granular, 


38 THE ACTINOMYCETES, Vol. II 


covered to a considerable extent with white 
aerial mycelium; piled up in center, discrete 
colonies at margin. 

Gelatin: Few flakes. No liquefaction. 

Milk: Heavy yellow growth attached to 
walls; solid coagulum in | month. 

Nutrient broth: Surface colonies cream- 
colored, scale-like; abundant,  flocculent 
bottom growth. 

Source: Infected rabbits. 

Remarks: Description given after Erikson 
(1935) 


53. Nocardia transvalensis Pijper and 
Pullinger, 1927 (Pijper, A. and Pullinger, 
B. D. J. Trop. Med. Hyg. 30: 153,(1927). 

Morphology: Initial growth made up of 
unicellular hyphae, the central branch being 
frequently broader and showing’ dense 
granular refractile contents. Aerial mycelium 
white, forming straight hyphae, in some 
cases becoming clustered into irregular 
spikes. Colorless drops are exuded and a pink 
coloration is produced in the densest part of 
the growth on synthetic glycerol agar. 
Angular branching with division of substrate 
filaments. Aerial hyphae irregularly seg- 
mented. Acid-fast. 

Glycerol nitrate agar: Growth in form of 
small, pink coiled masses. Aerial mycelium 
thin, white. 

Nutrient agar: No growth. 

Glucose nutrient agar: Colonies raised, 
granular, pink. Aerial mycelium white. 

Potato: Growth dry, raised, convoluted, 
pink. Aerial mycelium white. 

Gelatin: Growth poor, in form of a few 
irregular, colorless flakes. No liquefaction. 

Milk: No change. 

Starch: Not hydrolyzed. 

vee 


irregularly raised, coiled, dull pink mass. 


medium: Growth in form of small, 


Source: A case of mycetoma of the foot, in 
South Africa. 

Pathogenicity: To guinea pigs. 

Remarks: According to Gonzalez Ochoa 


and Sandoval (1956), N. transvalensis is ¢ 


synonym of NV. brasiliensis. 


54. Nocardia turbata Erikson, 1954 (Erik- 


son, D. J. Gen. Microbiol. 11: 198-208, 
1954). 
Morphology: Typical actinomycete, pro- 


ducing a fine mycelium composed of slender 
filaments, 0.1 « in diameter, which fragment 
into rods and coccoid cells. Under appro- 
priate conditions, many cells are motile. 
Nonacid-fast. 

Agar media: Growth good. Colonies small, 
0.1 to 2.0 uw. Initially colorless, later pro- 
ducing a yellow-green pigment on nutrient 
agar. Pigment production favored by free 
air supply, suppressed by acid reaction. 

Broth cultures: Turbid when young; sedi- 
mentation of cells later, when pellicle and 
clarification of medium produced. 

Acid production: Positive with glucose, 
sucrose, maltose, lactose, galactose, xylose, 
arabinose, glycerol, starch; negative with 
mannitol, raffinose, rhamnose, 
dulcitol (using a casein hydrolysate me- 
dium). 

Oxygen demand: Aerobic. 

Optimum temperature: 20-30°C. 

Nitrate: Utilized. 

Gelatin: No hydrolysis, except in presence 
of peptone (slowly). 

Paraffin utilization: Negative. 


sorbitol, 


Habitat: Probably soil. 


55. Nocardia uniformis Marton and 
Szabo, 1959 (Marton, M. and Szabo, I. 
Acta Microbiol. Acad. Sci. Hung. 6: 131- 


135, 1959). 

Morphology: The filaments of the sub- 
strate mycelium rapidly break up into rods 
and less frequently into coccoid bodies. The 
size of these forms is 0.7 to 1.1 uw by 1.1 to 
4.0 uw. In old cultures, swollen, club- or bottle- 
shaped forms appear. The hyphae of the 
aerial mycelium are 


slightly developed 


straight or waved, nonseptate, and contain 


THE GENUS NOCARDIA 09 


oval oidiospores. The mycelium is gram- 
positive and is not acid-fast. 

Agar media: The strains give nonbutyrous 
colonies growing into the agar, with moder- 
ately striated dull surface covered with 
shghtly developed white powder-like aerial 
mycelium. The color of the colonies is a 
constant yellowish-orange; it turns 
red or yellow; no soluble pigment is pro- 
duced. In liquid synthetic media a surface 
pellicle resembling agar colonies is formed. 

Gelatin: No liquefaction. 

Milk: No coagulation; no peptonization. 

Sugar inversion: None. 

Starch hydrolysis: None. 

Nitrate reduction: Rapid. 

Paraffin utilization: Shght or none. 

Optimum temperature: 14-37°C. 

Carbon utilization: Does not utilize man- 
nose, dextrin, inulin. 

Habitat: Deep layers B; horizon of saline 


never 


soils. 


56. Nocardia wpcottit (Erikson, 1935) 
Waksman (Gibson, A. G. J. Pathol. Bac- 
teriol. 23: 357, 1920; Erikson, D. Med. Re- 
search Council Spec. Rept. Ser. 203: 22-23, 
1935). 

Morphology: Growth forms long, straight 
filaments, much interwoven and ramified. 
Aerial mycelium slight, transient, slightly 
acid-fast. 

Glycerol Colonies small, 


nitrate agar: 


round, cream-colored, glistening; heavy 
texture, margins submerged. Later, growth 
very much convoluted and raised, broad 
submerged margin; medium becomes slightly 


reddish. 


Calcium malate agar: Growth limited, 
colorless, membranous, with undulating 


margin. 

Nutrient agar: Colonies smooth, shining, 
round, cream-colored; margin submerged. 
Aerial mycelium scant, white. Later, colonies 
are large with greenish tinge; very sparse 
aerial mycelium gradually disappears. 

Glucose nutrient agar: Colonies smooth, 


round, cream-colored; margin depressed, 
centers elevated, hollow on reverse side; 
later a coherent membranous growth, yellow- 
ish. 

Potato agar: Growth poor, in form of 
small, blister Medium 
shghtly discolored. 

Egg medium: Colonies round, flat, color- 
less, seale-like, some marked by concentric 
rings and slightly hollowed in center. Growth 
becomes yellow-brown. 

Blood agar: Colonies large, drab, heavily 
textured. No aerial mycelium. No hemolysis. 

Gelatin: Growth abundant, flocculent, 
cream-colored on surface. Gradual liquefac- 
tion. 

Source: From the spleen in a 
acholuric jaundice. 


colorless, colonies. 


case of 


57. Nocardia vacciniti Demarec and Smith, 
1952 (Demarec, J. B. and Smith, N. R. 
Phytopathology 42: 249-252, 1952). 

Morphology: Growth in form of rods and 
filaments, 0.4 u to 0.8 uw in diameter; granular 
appearance when stained; eventually break- 
ing up into bacillary forms. Few cells acid- 
fast. Fat demonstrated by staining with 
Sudan black B. 

Sucrose nitrate agar: Growth scant, gray. 

Nutrient agar: Growth poor, slow, granu- 
lar, gray, sometimes pinkish in old cultures. 

Gelatin: Growth dry, ribbon-like. No 
liquefaction. 

Starch nutrient agar: Growth dry, ribbon- 
like, pinkish to orange. Hydrolysis of starch 
positive. 

Potato: Growth slow, spreading, raised, 
oray. 

Milk: 
orange spots. No peptonization. 

Nitrate reduction: Positive. 

Carbon utilization: With ammonia as the 


Growth dry, raised, gray with 


source of nitrogen, acid formed from glucose, 
sucrose, glycerol, and mannitol; reactions 
variable with arabinose and xylose; no 
growth on lactose or sorbitol. 


Paraffin: Utilized. 


60 THE ACTINOMYCETES, Vol. II 


25-28°C; 


scant at 


Temperature: Growth best at 
inhibited at 32°C; none or very 
SAGE 

Antibiotic activity: None. 

Habitat: Causes formation of bud-prolifer- 
ating galls on blueberry plants. 

Type culture: ATCC 11,092. 


58. Nocardia variabilis (Cohn, 1913) Waks- 
man (Cohn, T. Centr. Bakteriol. Parasitenk. 
Orig. 70: 290-306, 1913). 

Morphology: Cells initially filamentous, 
breaking up into rods and cocci. Nonacid- 
fast. 

Agar media: Colonies round, smooth and 
lustrous, sometimes nodular; light brownish 
in color to orange-yellow. Colonies attached 
fast to the agar and partly removed with 
some effort. 

Gelatin: Growth orange-yellow. No lique- 
faction. 

Milk: Surface pellicle gradually becoming 
light orange. No coagulation; no peptoniza- 
tion. 

Broth: Colorless surface pellicle, readily 
dropping to bottom. Medium remains clear. 

Temperature: Optimum 387°C; — good 
erowth at 42°C; weak growth at 45°C. 

Potato: Growth thin, colorless, becoming 
in time yellow to orange-red; finally brown. 

Blood media: No hemolysis. 

Oxygen demand: Markedly aerobic. 

Habitat: Isolated from bladder of cystitis 
‘vases in man. Pathogenic to guinea pigs. 

Remarks: Said to be similar to A. ochroleu- 
cus, A. ochraceus, and A. carneus of Neukireh 
(1902). According to Krassilnikov (1949), it 
is closely related to NV. africana. 


59. Nocardia viridis (Krassilnikoyv, 1938) 
Waksman and Henrici, 1948 (Krassilnikov, 


N: A. Bull. Acad. Sci. USSR2 Nomi: 139; 
1938; Guide to the identification of bacteria 
and actinomycetes. Moskau, 1949). 

Morphology: Growth dark green in color. 
Colonies of doughy consistency on certain 
media (wort agar, potato), and compact on 
others (nutrient). Pigment insoluble in 
medium and in organic solvents. On protein 
media, cells develop to form a thin mycelium 
without visible walls. Cells often 
branching, 0.7 to 0.8 uw in diameter, with 
cross wall. After 5 to 7 days the cells break 
up into rods 5 to 15 w long. Coeei not ob- 
served. Cells multiply by fission, seldom by 
budding. No aerial mycelium. Gram-posi- 
tive. Not acid-fast. 

Nutrient agar: Growth compact. Thin 
mycelium produced. 

Potato: Growth rough, much folded. 

Gelatin liquefaction: Slow or none. 

Milk: No coagulation; no peptonization; 
some reports of positive peptonization. 

Starch: Not hydrolyzed. Spalla (1939) re- 
ported positive hydrolysis. 

Sucrose: Not inverted. 

Nitrate reduction: None. 

Paraffin and fats: Growth good; less on 
wax. 

Cellulose: No growth. 

Habitat: Soil. 

Davis and Freer (1960) described as a new 
species NV. salivae, an aerobic actinomycete 
isolated regularly from the human mouth. 
Strains of this species are characterized by 


cross 


their saccharolytic power, which thus distin- 
guishes them from the typical soil nocardias 
(see also von Magnus, 1947; Howell ef al., 
1959). Hirsch (1960) described N. saturnea, 
an aerobic organism occurring in dust and 
capable of utilizing petroleum. 


Chapter 4 


Characterization of Streptomyces Species 


Important Characters to be Considered 


for Recognition of Species and Va- 
rieties of Streptomyces 


In the identification and characterization 


of Streptomyces species, the following char- 


acters should be considered: 


I 


Morphological properties: 


(a) Structure of substrate mycelium. 

(b) Nature and formation of aerial my- 
celium. 

Structure and branching of sporo- 
phores. 

(d) Size and shape of spores. 

(e) Surface of spores. 

Cultural properties on various media: 
(a) Growth characteristics. 

(b) Development of aerial mycelium. 

(c) Color of aerial and substrate my- 


(¢) 


celium. 

Biochemical properties: 

(a) Production of soluble pigments in 
organic and in inorganic media. 

(b) Utilization of carbon sources. 

(ec) Starch hydrolysis. 

(d) Sucrose inversion. 

(e) Cellulose decomposition. 

(f) Proteolytic activities: liquefaction of 

gelatin, blood serum, and casein; co- 

agulation and peptonization of milk. 

Utilization of nitrogenous com- 
pounds. 

(h) Formation of oxidases: tyrosinase 
and laccase. 

(1) Reductases: nitrate reductase, sul- 
fate reductase. 


o> Or 


ints 


In characterizing 


(}) Formation of antibiotics and vita- 
mins. 

(k) Formation of H»S in peptone-iron 
agar. 

Sensitivity to antibiotics: 

(a) Sensitivity to pure antibiotic prep- 
arations. 

(b) Phenomena’ of  “‘cross-resistance”’ 
and ‘‘cross-sensitivity”’ on artificial 
media. 

Sensitivity to phages. 

Serological reactions. 

Chemical composition. 

Ecological properties. 

Genetic relationships. 

Age of culture. Information should be 

submitted concerning the age of the cul- 

ture when the particular properties were 
studied and the manner in which the 
culture has been kept in the laboratory. 

Type cultures. The culture should be 

deposited in a recognized collection and 

the assigned number reported. Every 
possible means for preservation of the 
culture should be used. Preservation of 
strains by lyophilization, soil culture, 
mineral oil seals on active slants, or 
storage in deep freeze is believed to re- 
duce physiological changes to a mini- 
mum. With the lyophilization technique 
within the reach of even the small lab- 
oratory, there is no excuse for an inves- 
tigator, particularly one publishing on 
designated strains, to ‘‘lose’’ his strains. 


Streptomyces species, 


only certain media should be used and well— 


62 THE ACTINOMYCETES, Vol. II 


defined conditions of growth recognized. Un- 
necessary media and nonessential details had 
better be left out to avoid cumbersome de- 
scriptions and nonduplicable characteris- 
tics that may apply to varieties or strains 
rather than to species. A larger number of 
media and more detailed descriptions may 
not only fail to give additional information 
but may complicate the description of the 
species to such an extent as to render the 
identification of freshly isolated cultures 
difficult. 

The composition of the media is usually 
given first consideration for descriptive pur- 
poses. According to Waksman (1958), Shi- 
nobu (1958), and others, these media should 
include: (a) at least three synthetic media, 
preferably sucrose-sodium-nitrate-salt or su- 
crose-ammonium-salt agar, glucose-.or glyc- 
erol-asparagine agar, and calcium malate or 
‘alecium citrate agar; (b) two or possibly 
three organic media, such as nutrient. (pep- 
tone-beef extract) agar, yeast extract-glucose 
agar, potato-glycerol-glutamate agar, or oat- 
meal agar; (c) three or four complex natural 
media, notably potato plugs, gelatin, and 
milk; (d) peptone-iron-yeast extract agar for 
HS production; (e) tyrosine medium for the 
tyrosinase reaction; and (f) a synthetic me- 
dium for carbohydrate utilization. 

Very few, if any, other media are required. 
Liquid media, with the exception of those 
previously listed, are better left out. 


Morphological Properties 


The method of study of the morphological 
properties of the streptomycetes would in- 
clude visual microscopic examination versus 
electron microscopic studies; direct exami- 
nation versus study of stained preparations; 
and hanging drop versus agar surface cul- 
tures. 


STRUCTURE OF SUBSTRATE MYCELIUM 


The substrate mycelium of a Streptomyces 
does not, as a rule, segment spontaneously 


into bacillary or coccoid forms. It produces 
leathery or tough-textured growth, remain- 
ing nonseptate and coherent even in old 
cultures. Although no true septa are ob- 
served in young cultures, it has recently been 
reported that older cultures show at least 
occasional septation. The compactness of 
this substrate growth is responsible for the 
fact that hquid media are always clear, un- 
less the culture has been subject to phage 
or lytic action. 


NATURE AND 
LIUM 


PROPERTIES OF AERIAL MYCE- 


The aerial mycelium is usually thicker 
than the substrate mycelium. While the 
morphology of the substrate mycelium is 
usually undifferentiated, the aerial mycelium 
of streptomycetes, under fixed conditions of 
culture, shows sufficient differentiation that 
a miscellaneous assortment of isolates can 
be segregated into a number of groups hav- 
ing like morphological characteristics. This 
is one of the most important criteria for 
classification in the genus Streptomyces. Sev- 
eral aspects relating to the aerial mycelium 
may be considered: 

a. Gross macroscopic appearance. The rela- 
tive abundance, structure (cottony, velvety, 
powdery), formation of rings or concentric 
zones, and pigmentation of the aerial my- 
celium are important diagnostic criteria. 

b. Microscopic properties. The microscopic 
structure of the aerial mycelium gives a clear 
picture of the morphology and reproductive 
structures of the organism. The hyphae may 
be long or short, with extensive or little 
branching. The branching may be simple or 
complex, monopodial or sympodial, broom- 
shaped or verticillate. The fruiting bodies or 
sporophores are short or long, occurring sin- 
gly, in clusters, or as verticils; they are 
straight, wavy, or spiral-forming. The spirals 
or coils are either long and open or short 
and compact. Spiral formation may take 
place on one medium and not on others. 


CHARACTERIZATION OF 


| 


i 


STREPTOMYCES SPECIES 63 


FiaureE 16. Schematic representation of different types of spirals produced by various Streptomyces 


species; the spirals range from long to short, from compact to irregular (Reproduced from: Shinobu, R. 
Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sei. 7, 1958). 


Before a culture is pronounced as forming 
no spirals, therefore, it must be grown on a 
variety of selective media that will allow 
optimum sporulation. Drechsler (1919) sug- 
gested use of the right-hand or left-hand 
curvature of the spirals as a diagnostic fea- 
ture, but 
composition of the medium (Ettlinger ef al., 
1958: Shinobu, 1958). Verticil formation is 


also an important characteristic of certain 


this, too, is influenced by the 


species; it can be simple or branching (pri- 
mary or secondary verticils), the branches 
being straight or forming spirals; but this 
property as well is influenced to some extent 
the medium. Al- 
though nocardiae may produce sporulating 


by the composition of 


aerial filaments, these are never spiral- 
shaped (lig. 16). 
Waksman (1940, 1950) divided the or- 


ganisms belonging to the genus Actinomyces 
(largely the forms now included in the genus 
Streptomyces) into the following five sub- 
groups on the basis of the structure of the 
sporulation hyphae. 

I. Straight sporulating hyphae, monopo- 


dial branching, never producing regular 
spirals. 

Il. Spore-bearing hyphae arranged in clus- 
ters, or broom-shaped arising from 
compression of the sporophores. 

III. Spiral formation in aerial mycelium; 
long, open spirals. 

IV. Spiral formation in aerial mycelium; 
short, compact spirals. 

V. Spore-bearing hyphae arranged on my- 
celium in whorls (verticils) or tufts. 

(1941, 1949) the 


genus Actinomyces (largely forms included 


Krassilnikoy divided 
in Streptomyces) on the basis of the following 
properties: (1) spiral forming versus straight 
sporophores; (2) alternate distribution of 
sporophores on aerial mycelium versus verti- 


) 


cil formation; (8) spherical versus oval 


spores; (4) colorless versus pigmented cul- 


tures; (5) white versus colored aerial my- 
celium; (6) saprophytes versus parasites. 

Aiso et al. (1948) divided the genus Strep- 
tomyces on the basis of the structure of the 
aerial mycelium into six types: 


I. Spirals not formed. 


64 THE ACTINOMYCETES, Vol. II 


F 


Piate I. Diagrammatie representation of the morphology of the sporophores of Streptomyces (Re- 
produced by special permission from Ettlinger et al. Arch. Mikrobiol. 31: 336, 1958). 
a. Sporophores produce straight branching verticils on sterile aerial hyphae; S. retdculi type. b. Sporo- 
phores produce open spirals as side branches on sterile aerial hyphae; S. purpurascens type. ¢. Sporo- 
phores produce on sterile aerial hyphae verticils with open, more or less regular spirals; S. nowrsei type. 
d. Sporophores formed as side branches on sterile aerial hyphae, straight or slightly wavy; S. phaeo- 
chromogenes. e. Sporophores produced as verticils on sterile aerial hyphae, with open, irregular spirals; 
S. echinatus. {. Sporophores monopodially branched, forming irregular open spirals at the end of long 
hyphae; S. lavendulae. g. Sporophores monopodially branched, with open, regular spirals; S. parvullus. 


CHARACTERIZATION OF STREPTOMYCES SPECIES 65 


mM n fe} Pp 


Prats II. Diagrammatic representation of the morphology of the sporophores of Streptomyces (Re- 
produced by special permission from Ettlinger ef al. Arch. Mikrobiol. 31: 337, 1958). 
h. Sporophores produce numerous short, monopodial branches on sterile hyphae; S. ramulosus. i. Sporo- 
phores sympodially branched, forming tufts upon short main axes; S. griseus. k. Sporophores produced 
upon a long, straight main axis, monopodially branched with frequent, regular spirals; S. fradiae. 1. 
Sporophores monopodially branched, straight or slightly wavy; S. antibioticus. m. Sporophores mono- 
podially branched along the whole axis with open, irregular spirals; S. erythraeus. n. Sporophores mono- 
podially branched, with narrow, compact spirals; S. violaceoniger. 0. Sporophores sympodially branched, 
in the form of trees with a long main axis; S. viridogenes. p. Sporophores monopodially branched, stiff 
and straight; S. venezuelae. 


69 THE ACTINOMYCETES, Vol. II 


1. Straight, very little branching. 
2. Wavy, abundant branching. 

II. Spirals formed. 

1. Spirals formed on the axis, irregu- 
lar branching. 

2. Spirals formed on branches in clus- 
ters. 

Verticils produced. 

1. Verticillate branches entangled like 
a net. 

2. Verticillate branches formed on both 
axis and branches, making primary 
and secondary verticils. 

Okami (1952) grouped the genus Strepto- 
myces on the basis of formation of aerial 
mycelium into the following types: 

I. Spirals not formed. 
1. Branches produced. 
2. Branches not produced. 

II. Spirals formed. 

1. Spiral form mostly compact. 
2. Spiral form mostly loose. 

Shinobu (1958) eriticized the systems of 
Aiso and Okami on the basis that insufficient 
attention was paid to the nature of the me- 
dium. In the system of Aiso et al. the distine- 
tion between | and 2 of each type appeared 
to him to be unclear, many forms belonging 
to an intermediate type. Okami’s system 
was considered as incomplete because the 


1 


formation of verticils was not taken into 
consideration. Waksman’s system was_ be- 
lieved to be comparatively better, but even 
this system was criticized because cluster or 
broom-shaped sporophore formation was not 
considered as a sufficient characteristic, and 
because a strain does not necessarily have 
only one kind of spiral, but usually forms 
various kinds of spirals which coexist (PI. IT). 

Hesseltine et al. (1954) and Pridham et al. 
(1958) considered the sporophore morphol- 
ogy as reasonably stable under definite nu- 


tritional requirements of the organisms. 
Several morphological groups were sug- 


gested. The components of each group were 
considered as suggesting a logical natural 
arrangement. The physiological data can be 


’ 


used to produce ‘“‘species’”? or ‘‘species- 
groups,” with morphology as a_ starting 
point. Seven morphological sections were 
created as subdivisions of the genus Strep- 
tomyces. However, this system as well was 
considered by Shinobu as having certain de- 
fects because (a) culture media for morpho- 
logical study were not examined thoroughly 
enough; and (b) some of the sections may 
often coexist In one strain. 

Shinobu (1958) emphasized the following 
morphological properties of the aerial my- 
celium: 

a. Outward appearance — of 
(powdery, cottony, leathery). 

b. Branching, especially the formation 
and nature of verticils. 

ce. Formation and nature of spirals. 

d. Formation and shape of spores. 

e. Thickness and length of mycelium. 

Shinobu examined in detail the various 
properties of the aerial mycelium, emphasiz- 
ing again the need for suitable synthetic 
media. He concluded that the nature of the 
aerial mycelium is one of the most important 
characteristics for taxonomic study, but that 
it should be considered in connection with 
composition of the medium. The aerial my- 
celium was classified into the following three 


mycelium 


groups, from the standpoint of branching 
and formation of spirals. 

Group I. Straight or wavy aerial my- 
celium, monopodial branching, never pro- 
ducing spirals or clusters. 

Group II. Spiral formation in the aerial 
mycelium; long or short; loose or compact; 
open or closed. 

Group III. Verticil or cluster formation 
in the aerial mycelium. 

The loss of ability to form aerial mycelium 
and sporogenous hyphae by certain Strepto- 
myces cultures, on the one hand, and the 
formation of aerial mycelium and_ sporo- 
phores by certain species and strains of 
Nocardia, on the other, led Bradley (1959) 
to question the distinction between these 
two genera. We have here simply another 


CHARACTERIZATION OF STREPTOMYCES SPECIES 67 


vase of natural overlapping between man- 
made concepts of genera and species or the 
improper labeling of cultures. Gordon and 
Mihm (1957) emphasized that it is easy to 
understand how a culture of NV. 
that formed acid-fast coecobaecilli, rods, and 
short filaments and growth 
heavy, finely to coarsely wrinkled, cream- 
colored to orange, and without noticeable 
aerial hyphae, could be mistaken for a J yco- 
bacterium. A culture of V. asteroides, how- 
ever, that produced nonacid-fast, long, tan- 
gled filaments and a cream-colored, pale 
yellow, or beige growth thickly covered with 
whitish aerial hyphae, could just as easily be 
accepted as a Streptomyces. 

Numerous other studies have been made 


asteroides 


whose was 


of the micromorphology of the various spe- 
cies and groups of Streptomyces, as in the 
work of Burkholder et al. (1954), Hesseltine 
et al. (1954), Ettlinger et al. (1958), Flaig 
and Kutzner (1960), and others. 

c. Spores. The spores, also called conidia, 
produced from, or in, certain hyphae of the 
aerial mycelium, or the ‘‘sporogenous hy- 
phae,”” may be oblong, oval, or spherical. 
Krassilnikov (1949) attached great impor- 
ance to this character, as determined by the 
light microscope, as a diagnostic feature. 
Kriss et al. (1945) were the first to use the 
electron microscope for study of spores of 
Streptomyces. This was followed by the work 
of Carvajal (1946); Kiister (1953); Flaig et 
al. (1952, 1955, 1958); Baldacci and Grein 
(1955); Grein (1955); Vernon (1955); and 
others. Flaig et al. (1952) found that the 
spores of some strains had smooth surfaces 
while others had spiny surfaces. They later 
detected spores with hairy and warty sur- 
faces; the nature of the nitrogen source in- 
fluenced the appearance of the spore surface, 
organic nitrogen favoring spine formation. 
Kiister (1955) classified Streptomyces spores 
into two groups: (a) those producing a 
smooth surface and (b) those having a rough 
surface. Each of these groups was divided 


into three subgroups, based on shape of the 
spores. Thus there are spores with smooth 
surfaces, with spines, with hairs, or with 
warty protuberances, and spores that are 
globose, long-ovoid, and cylindrical. 

On the basis of a system of classification 
that they had outlined, Baldacci and Grein 
(1955) examined 50 strains of streptomycetes 
with the electron microscope. Three types 
of spores were recognized: (1) Oval, more or 
less transparent spores; these were either 
smooth or rough, the latter having a spiny 
or hairy surface; the spines were either short 
and thick or long and thin. (2) Round, 
opaque spores, usually smooth. (3) Poly- 
hedral spores, smooth and transparent, or 
shghtly curved, wrinkled, and opaque. The 
form of the spores was constant for the series 
in Baldacci’s system. It can hardly be used, 
however, as a species characteristic. A cor- 
relation was observed (Pridham, 1959) be- 
tween spore characteristics and sporophore 
morphology (Table 6). 

According to Preobrajenskaya et al. (1959, 
1960), strains within one species as a rule 
have a similar type of spore surface. Cultures 
with a white, yellow, greenish-yellow, yellow- 
gray, pink, or lilae mycelium have smooth 
spores; those with a bluish aerial mycelium 
have spiny and hairy spores, and species 
with a gray aerial mycelium have spores of 
all types. The diagnostic value of spore sur- 
face characteristics was found to be dissimi- 
lar for the various sections. The correlation 
between the gray and bluish species and the 
character of the surface of the spores was 
considered as insignificant. Tresner ef al. 
(1960) also emphasized the importance of 
spore surface in classifying species of Strep- 
tomyces; size and shape of spores of most 
species were considered of limited usefulness 
in taxonomic differentiation. 

Lechevalier and Tikhonienko (1959) re- 
ported that the spores of S. viridochromo- 
genes were mostly elongated and those of S. 


violaceus, spherical. The spines formed by 


68 THE ACTINOMYCETES, Vol. II 


TABLE 6 


Morphology of sporophores and spores of streptomycetes* (Pridham, 1959) 


eee AIREE Original strain no. Sporophore morphologyt BAN Ce 
griseus B-1598 Carvajal SL 842 Straight to flexuous (RF) Smooth-walled 
bikiniensis B-1049 Waksman 3515 Straight to flexuous (RE) Smooth-walled 
CaNesCus 2419 Com. Sol. 811.0 Straight to flexuous (RF) Smooth-walled 
venezuelac B-902 Gottheb 8-44 Straight to flexuous (RF) Smooth-walled 
cinnamonen- _B-1588 Okami 154-T3 Hooks and open loops (RA) Smooth-walled 
sis with many straight (RF) 
sporophores 
flaveolus B-1334 ATCC 3319 Hooks and open loops (aber- Spiny to hairy 
rant) (RA) 
albus B-1685 Waksman (ATCC Spirals (aberrant) (S) Smooth-walled 
618) 
hygroscopicus B-1865 NRRL isolate Spirals (8) Spiny 
chartreusts 2287 Upjohn K-180 Spirals (8) Spiny 


* Data taken from Carvajal, 1946; Vernon, 1955. All other data on spore morphology supplied by 
K. L. Jones. 
+ Sporophore morphology determined according to the methods of Pridham ef al., 1958. 


Fiaurp 17. Coremia formation by certain Streptomyces species, X 500; stained by Corti’s method 
(Courtesy of Dr. J. Giolitti, Milan, Italy). 


CHARACTERIZATION OF STREPTOMYCES SPECIES 69 


strains of both species differed cytologically. 
The spines of the first seemed to be part of 
the cell wall, whereas the spines of the sec- 
ond seemed to be very superficial, appearing 
only on the envelope. It was concluded that 
spine formation is a stable characteristic of 
the spores. The shape of the spores varied 
with the composition of the medium. It was 
suggested that complex organic media be 
avoided for spore study. 


COLONY STRUCTURE 


The nature of the Streptomyces colony 
growing on a standard agar plate has been 
considered as among the important criteria 
for characterizing and recognizing a particu- 
lar organism. One may question, however, 
the significance of this property in describing 
a species. The morphology of the colony, 
notably its general appearance, size, shape, 
and texture, can all be readily determined 
by superficial examination. Various other 
properties may be recognized from a study 
of the colony. Krainsky used the structure 
of the colony, especially its size and shape, 
as one of the major diagnostic criteria. 

The superficial examination of gross col- 
ony structure, particularly its texture, can 
be of some help. Pridham* and others have 
noted the following very general correlations: 

1. Straight to flexuous cultures generally 
are flat with a velvety, granular, or powdery 
texture. 

2. Loop cultures generally are flat with a 
velvety to slightly granular texture. 

3. Spiral cultures generally are elevated 
with a somewhat floccose texture. Occasion- 
ally, spiral cultures that are flat with a vel- 
vety or granular texture may be seen. In 
spiral cultures that are somewhat floccose 
the sporulating aerial mycelium often con- 
sists of long sterile hyphae with sporophores 
branched oppositely, singly, or sometimes in 
an apparent verticillate fashion. 


* Personal communication. 


FicureE 18. Sclerotium formation in Strepto- 
myces (Prepared by H. Lechevalier, Institute of 
Microbiology). 


Figure 19. Sclerotium in species of Strepto- 
myces, designated as new genus Chainia by Thir- 
umalachar (Prepared by H. Lechevalier, Institute 
of Microbiology). 


4. Verticillate cultures generally are ele- 
vated and floccose. Aberrant verticillate cul- 
tures generally are flat with a velvety tex- 
ture. 


70 THE ACTINOMYCETES, Vol. II 


Fiaure 20. Sclerotium in species of Strepto- 
myces, designated as new genus Chainia (Prepared 
by H. Lechevalier, Institute of Microbiology). 

According to Krassilnikov (1955, 1959, 
1960), the nature of the sporophore is a per- 
manent property, being straight in the S. 
globisporus group, spiral-shaped in the S. 
coelicolor and S. violaceus groups. The same 
constancy is true of the shape of the spores, 
cylindrical versus oval or spherical, and of 
the manner of spore formation, namely, frag- 
mentation versus segmentation. Branching 
of the sporophores, namely, vertical forma- 
tion versus monopodial branching, 1s also a 
constant, although a variable, property. No 
single property, however, 1s sufficient to 
characterize species. Coremia formation 
(Fig. 17) is of no taxonomic significance; 
however, production of sclerotia is believed 
to be a constant property, in agreement with 
Thirumalachar (1955), but not with Gattani 
(1957), who denied its significance (Tigs. 
18-20). 


Cultural and Biochemical Characteristics 


FORMATION OF PIGMENTS 


Among the cultural properties of strepto- 
mycetes, the color of the substrate growth 
of the aerial mycelium and the spores and 
the formation of soluble pigments in organic 
and synthetic media play a major role in 
characterizing species. This fact is amply 
illustrated by the numerous specific epithets 
referring to color that have been used to 
designate various organisms. Unfortunately, 
color characteristics vary greatly with age 
of the culture, composition of the medium, 
temperature of incubation, and nature of the 
inoculum. 

Before the introduction of synthetic me- 
dia, it was a common practice to divide the 
actinomycetes into two groups: (a) colorless 
or nonchromogenic, and (b) pigment-produce- 
ing or chromogenic forms. The latter com- 
prised those organisms that produced deep 
brown to black diffusible pigments when 
grown on proteinaceous media. With the in- 
troduction of synthetic media, 1t came to be 
recognized that different organisms are able 
to produce a great variety of pigments, rang- 
ing from red to blue and from orange and 
yellow to brown and black. Some are single 
pigments, and others comprise two or more 
constituent pigments. Some are water-sol- 
uble and others are water-insoluble, as shown 
in detail in Chapter 13 of Volume I. The 
presence of oxygen is essential for pigment 
formation. The pH of the medium greatly 
affects the nature of the pigments, both in- 
soluble and soluble. 

When cultures are grown on optimum 
sporulation media, the pigmentation of the 
spores is highly significant; 1t may be ob- 
served at an early growth stage, at maturity, 
or only in old cultures, since changes in color 
may occur with age of culture. 

The formation of deep brown to black pig- 
ments on organic media containing proteins 


CHARACTERIZATION OF 


and protein derivatives, notably the amino 
acid tyrosine, 1s an important species charac- 
teristic. Certain species may produce only 
faint brown soluble pigments on organic 
media, as well as on synthetic media. Differ- 
ent cultures, especially on continued cultiva- 
ion artificial media, will 
variation in pigment production. 

Since about one third of all species of 


on show great 


Streptomyces now recognized are melanin- 
positive (Waksman, 1919; Skinner, 1938), 
and since this property has been utilized ex- 
tensively in the classification of actinomy- 
cetes, a knowledge of this reaction is of great 
importance. Gasperini (1891) first utilized 
this property in dividing the aerobic Actino- 
myces into A. chromogenus and A. albus. It 
was later recognized, however, that melanin 
production is characteristic of a large number 
of species, including such important forms 
as the plant pathogen S. scabies. 

Beijerinck (1900, 1911, 1913) designated 
as “melanin” the dark pigment produced by 
A. chromogenus from peptone, although this 
organism did not always produce the pig- 
ment from tyrosine. He considered the pig- 
ment as a catabolic product of the organic 
nitrogen. 

Lehmann and Sano (1908) first suggested 
the expression “‘tyrosinase reaction.”’? They 
used for their studies a tyrosine-containing 
medium, melanin being known to be an oxi- 
dation product of tyrosine. Waksman (1916, 
1919, 
that the production of a soluble dark pig- 
ment on beef-peptone agar is due solely to 
this reaction. Gelatin, containing no tyro- 


1920) expressed considerable doubt 


sine, gives the characteristic pigmentation. 
Some species producing a typical dark pig- 
ment on the beef-peptone agar may fail to 
do so on synthetic media containing tyrosine. 

Skinner (1938) recognized a difference be- 
tween the dark pigment produced in peptone 
media and not in tyrosine-containing syn- 
thetic media and the black pigment pro- 


STREPTOMYCES SPECIES rial 


duced in tyrosine (or protein containing 
tyrosine) media. The tyrosinase reaction 
was, therefore, considered to be the proper 
one 

Shinobu (1958) attached great importance 
to the “tyrosinase reaction”’ in the species 
characterization of Streptomyces. Ettlinger 
et al. (1958) also recognized the difference 
between melanin formation and the tyrosi- 
In line with the of 


Beijerinck, Waksman, and Ettlinger et al., 


nase reaction. ideas 
recognition will be given here to melanin 
formation rather than to the tyrosinase re- 
action. 

The formation of yellow, red, blue, green, 
and other soluble pigments is also highly 
characteristic of the species growing on syn- 
thetic media. There is considerable variation 
in the intensity of these pigments, depending 
upon the strain of organism. In view of the 
fact that color standards are not always 
available, Lindenbein (1952) suggested a 
series of color designations which are simple 
and convenient. This system in a modified 
form is given in Appendix I. 

Pigment formation is considered by Kras- 
sinikovy (1960) as a constant specific prop- 
erty, although the nature of the pigments 
varies with the composition of the medium. 
The color of the aerial mycelium is not con- 
sidered as constant and is greatly influenced 
by the composition of the medium (see also 
Conn and Conn, 1941). 

The variability in pigmentation of differ- 
ent strains of S. aureofaciens was studied in 
detail by Duggar et al. (1954) and Backus 
et al. (1954), and is illustrated in Table 7. 


UTILIZATION OF CARBON SOURCES 


The ability of different species of actino- 
mycetes to utilize as sources of carbon and 
energy various organic substances, such as 
ot 


acids, fats, and amino compounds, can be of 


carbohydrates, alcohols, salts organic 


considerable diagnostic value. These studies 


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CHARACTERIZATION OF 


the early work of Waksman 
(1919), who employed a synthetic solution 
to which he added various carbon- or ni- 


trogen-containing compounds. Liquid sub- 


date from 


strata were employed and cultures were 
incubated under static conditions. The use 
of static hquid substrata was later found to 
give misleading results. In some cases, uni- 
form inoculum distribution is not achieved 
unless considerable care is taken. Numerous 
studies (Pridham and Gottlieb, 1948; Bene- 
dict et al., 


1955; ete.) indicate that solid 


substrates and different basal media 
later used (Table 8). 


Hata et al. (1953) found a correlation be- 


were 


tween the groups and types of organisms 
established on the basis of carbon utilization 
and their antistreptomytic and antibae- 
terial spectra. 

Zahner and Ettlinger (1957) did not at- 
tach major significance to the utilization of 
carbon sources for characterizing species of 
Streptomyces. They suggested that such in 
formation be combined with other criteria. 
None of the 125 cultures they studied could 
use dulcitol, for example. The best carbon 
sources for characterizing Streptomyces spe- 
cies were found to be raffinose, /-xylose, d- 
fructose, /-arabinose, and d-mannitol. Gor- 
don and Mihm (1959) considered as a species 
the 
malate, propionate, pyruvate, and succinate. 


characteristic utilization of acetate, 

None of the actinomycetes produce gas. 
Some are able to form acid, such as lactic, 
from certain carbon sources. Gordon and 
Smith (1954) used acid production from lae- 
tose, maltose, xylose, and mannose as one 
of the criteria for differentiating Nocardia 
and Streptomyces species. Gordon and Mihm 
(1959) later suggested for species separation 
the use of acid formation from glycerol, glu- 
cose, arabinose, erythritol, inositol, lactose, 
maltose, mannitol, and certain other carbon 
sources. 


Carbon source utilization by 12 natural 


STREPTOMYCES SPECIES 


TABLE 8 


variant strains of S. aureofaciens 
(Backus ef al., 1954) 


No apparent 


utilization by 


Utilization by 
all variants 


Utilization variable with strain 


any variant Posi- | Nega- 
tive tive 
Sodium Glucose Galactose 11 i 
acetate* Sucrose Sodium 10 2 
Sorbose Maltose citrate* 
Glycine Starches Levulose 9 3 
Mannitol Dextrin Mannose 9 3 
Arabinose Trehalose Lactose i) 3 
Glycerol Magne- 7 5 
| Sodium sium lac- 
succl- tate* 
nate* Xylose 6 6 
Inulin 


* Used at 0.4 per cent level, all others at 1 per 
cent. 
PROTEOLYTIC ACTIVITIES 

Among the proteolytic activities of diag- 
nostic value in separating genera, liquefac- 
tion of gelatin, hydrolysis of casein, and pep- 
tonization of milk are very important. 

Species of Nocardia effect little, if any, 
liquefaction of gelatin, whereas most species 
of Streptomyces bring about liquefaction. 
The rapidity of liquefaction varies greatly. 
Some species show strong activity, and 
others give only limited liquefaction. This 
property, as well as milk peptonization, when 
combined with the ability of the species to 
produce brown to black pigments, provides 
significant criteria for species characteriza- 
tion. 

In a study of 477 cultures of Streptomyces, 
Stapp (1953) found only one that did not 
liquefy gelatin. Detailed studies on proteo- 
lytic activities of actinomycetes are found 
in the work of Waksman (1919), Jensen 
(1930), Gordon and Smith (1955), and Kutz- 
ner (1956), as well as in Vol. 1, pp. 183-186. 
Waksman (1919) reported that of 35 cultures 
tested, 33 liquefied gelatin more or less rap- 


74 THE ACTINOMYCETES, Vol. II 


idly; when these tests were repeated three 
times, considerable variation in the degree 
of liquefaction was obtained. IXutzner (1956) 
kept gelatin cultures for 31 days; only four of 
210 failed to liquefy the gelatin. Reports of 
inability to liquefy gelatin or coagulate or 
peptonize milk of certain species may often 
be questioned. Repeated tests with different 
inocula might have shown different results. 
Gordon and Smith (1955) suggested casein 
hydrolysis as one criterion for the separation 
of Streptomyces strains from those of Nocar- 
dia. 

Stapp (1953) reported further that in his 
collection 18 cultures brought about coagula- 
tion of milk without subsequent peptoniza- 
tion, 431 caused coagulation and peptoniza- 
tion, and 19 caused peptonization without 
previous coagulation. A few cultures ocea- 
sionally are found that cause no coagulation 
or peptonization. One wonders whether re- 
peated tests with different inocula might 
show different results. 


REDUCING PROPERTIES 


The reduction of nitrate to nitrite has 
been universally used among the criteria for 
species differentiation. In view, however, of 
the influence of nutritional factors upon this 
reaction, and its quantitative rather than 
qualitative nature, its significance in species 
characterization may be questioned. 

Proteolysis, starch hydrolysis, sucrose in- 
version, cellulose utilization, and nitrate re- 
duction were said (IXrassiinikov, 1960) to be 
characteristic of all actinomycetes 
and to have, therefore, no taxonomic signifi- 


almost 


cance. Sugar assimilation was considered, 
however, as a more or less constant property. 


UTILIZATION OF NITROGEN SOURCES 

As a rule, utilization of nitrogenous com- 
pounds has not been considered important 
for species characterization. Shinobu (1958) 
considered the utilization of urea, creatinine, 
and certain amino acids as of some impor- 


tance in species characterization. Gordon 
and Mihm (1959) suggested that the ability 
to attack casein, tyrosine, or xanthine can 
be considered of some significance in charac- 
terizing species. 

The use of hydrogen sulfide production as 
a taxonomic implementation in the differen- 
tiation of Streptomyces species has been sug- 
gested by Pridham (1948). Tresner and 
Danga (1958) later modified the peptone- 
iron agar medium. More than 900 strains 
belonging to one or another of 94 species 
were tested. There was a marked difference 
in response from strain to strain within a 
species; for example, 98 per cent of 227 
strains of S. hygroscopicus were negative; 99 
per cent of 112 isolates of S. lavendulae were 
positive. When employed in conjunction 
with other physiological, cultural, and mor- 
phological criteria, hydrogen sulfide produc- 
tion was said to give promise as an aid in 
the systematics of the genus Streptomyces. 


Sensitivity to Antibiotics 


Actinomycetes, especially species of Strep- 
tomyces, have been found in recent years to 
produce a series of highly valuable chemical 
substances, notably, antibiotics. This prop- 
erty has come to be considered as highly 
characteristic of a given species. The fact 
that a large proportion of all the cultures of 
Streptomyces isolated from natural 
strates show some degree of inhibition of 
growth of other microorganisms, when tested 


sub- 


on suitable media, suggested the ability to 
form antibiotics to be of potential diagnostic 
value. It is a question whether one is a 
“Jumper” or a ‘splitter’? when one regards 
the ability to produce an antibiotic as a 
species rather than a strain characteristic. 
Certain antagonistic strains belonging to the 
S. griseus group, for example, are able to 
produce various streptomycins and eyclo- 
heximide. Others may form various actino- 
mycins, grisein, streptocin, or candicidin. 

It has been suggested that because the 


CHARACTERIZATION OF STREPTOMYCES SPECIES 


growth of homologous strains of an organism 
is less inhibited than that of heterologous 
forms, added weight could be given to the 
potential diagnostic value of antibiotic pro- 
duction. The application of the concept of 
antibiosis as a major characteristic for the 
speciation of actinomycetes is not generally 
accepted, since the metabolism of these or- 
ganisms is too complicated to give sharp 
lines of autoinhibition. At most, it can be a 
varietal rather than a species characteristic. 

Krassilnikov (1950, 1958, 1960a, 1960b) 
tended to overemphasize the importance of 
antibiotics in species characterization of ac- 
tinomycetes. He made two unjustified as- 
sumptions: (a) every species synthesizes only 
one particular antibiotic, (b) antibiotics do 
not inhibit the growth of organisms belong- 
ing to the species producing such antibiotics. 
Many species and even individual cultures 
are able to form a variety of different anti- 
biotics; on the other hand, the same anti- 
biotic may be produced by different organ- 
isms. The growth of an organism may in 
some instances actually be inhibited by its 
own antibiotic, as with S. fradiae and neo- 
mycin. Finally, a single culture may produce 
mutants which either have lost the ability 
to form a particular antibiotic or have gained 
the ability to synthesize a totally different 
antibiotic. It is somewhat dangerous to use 
assumptions and generalizations as the basis 
for species characterization. The importance 
of considering antibiotic formation in the 
systematization of actinomycetes has also 
been emphasized by Gause (1955). 


Actinophage Sensitivity 


During the last 10 years several attempts 
have been made to determine whether 
“phage-typing” of actinomycetes might be 
of some help in identifying unknown isolates. 
The results obtained point to several facts 
which must be kept in mind if one tries to 
use this procedure for characterizing and 
Classifying Streptomyces species. 


~J 
) 


1. Actinophages vary greatly in their host 
ranges. 

a. Most which 
tested against a large number of organisms 
proved to be polyvalent; that is, they lyse 
cultures that belong to different species or 
even genera (different according to our pres- 
ent species concept, which is based on the 
system presented here). The data presented 
by Bradley and Anderson (1958) might serve 
as an illustration (Table 45, Volume I). The 
activity of some phages upon members of 
the genera Streptomyces and Nocardia led 
these workers to question the validity of 
separating these two genera, which have ac- 
tually been placed in two different families 
within the order Actinomycetales. Activity 
of a polyvalent Streptomyces phage on two 
Nocardia species was also found by Gilmour 
et al. (1959). In a study carried out by St. 
Clair and McCoy (1959), however, nine 
phages which proved to be polyvalent 
against several Streptomyces species failed to 
attack any of the four Nocardia species 
tested. The polyvalent character of other 
phages tested against other species has been 
shown also by other investigators (Hoehn, 
1949; Chang, 1953, Rautenstein and Kofa- 
nova, 1957; Gause et al., 1957; Mach, 1958; 
Shirling, 1959a, b; Kutzner and Waksman, 


actinophages were 


1959a; Kutzner, 1960). Obviously, therefore, 
a phage characterized by a wide host range 
is usually of little value in species differ- 
entiation, unless one is inclined to be a 
“Jumper”? who demonstrates by the use of 
a polyvalent phage that he is right and the 
“splitters”’ are wrong. 

b. Some phages have been found to be 
specific, causing the lysis of strains of only 
a few species or of only certain strains of one 
species. In the latter case one might be justi- 
fied in doubting the uniformity of the spe- 
cies rather than in considering the phage as 
“‘superspecific.”’ This seems to be definitely 


true of the species ‘‘S. griseus,’’ various 


76 THE ACTINOMYCETES, Vol. II 


strains of which show a very different sensi- 
tivity pattern against certain phages. 

It is true, likewise, of the separation of 
streptomycin-producing strains from grisein- 
producers and other members of the former 
S. griseus, which is now regarded as a species 
group rather than a single species (Waks- 
man, 1959). There have been various reports 
concerning phages which are active upon 
streptomycin-producing strains, but do not 
attack grisein-producers or nonantibiotic- 
forming cultures (Woodruff et al., 1947; 
Waksman et al., 1947; Reilly et al., 1947; 
Waksman and Harris, 1949; Hoehn, 1949; 
Carvajal, 1953; Burkholder eé al., 1954). 
Some streptomycin-producing cultures have 
been found, however, that are resistant to 
these specific phages (Okami, 1950; Carva- 
jal, 1953; Kutzner, 1960). Other phages have 
been discovered that are specific against S. 
coelicolor (Kutzner and Waksman, 1959a; 
Kutzner, 1960), S. lavendulae (Gause et al., 
1957; Shirling, 1959), and SS. olzvaceus (KXha- 
vina and Rautenstein, 1958). 

2. No general conclusion can be drawn 
from the spectrum of a polyvalent phage in 
regard to relationships between lysed strains. 
However, a polyvalent phage can be useful 
in taxonomic studies if it shows specificity 
within a particular group of organisms that 
are very similar in their other properties 
and therefore hardly distinguishable. Fur- 
ther, testing several polyvalent phages might 
result in typical sensitivity patterns of the 
organisms which might be of some value in 
recognizing whether one has to do with 
closely related or unrelated organisms. 

3. Actinophages vary greatly when tested 
against numerous strains. In some cases dif- 
ferences in plaque counts might be due to 
host range mutants which are present at a 
concentration of 10° to 10° particles. These 
mutants would attack a ‘‘new host”’ resist - 
ant to the parent phage, as shown by Welsch 
(1954, 1957) and Welsch et al. (1957). In 
numerous other cases, however, the devel- 


opment of host range mutants cannot ex- 
plain the wide host range, and the phages 
retain their polyvalent nature even after 
several serial passages on heterologous hosts 
(Chang, 1953; Shirling, 1959a; Bradley, 
1959; Gilmour et al., 1959). It is necessary 
to carry out phage tests with different dilu- 
tions of the original phage preparation, 
which should contain about 107 to 10° par- 
ticles per milliliter. 

4. A survey of the literature shows that 
almost every investigator uses a different 
medium for phage typing. The methods 
comprise either spot tests or single plaque 
counts. In a comparative study of different 
media for phage typing, Kutzner (1960) 
found that some phages gave similar plaque 
counts on a variety of media. However, 
counts of phages that formed tiny plaques 
were found to be quite dependent on media 
composition. Inorganic salt content of media 
was found to influence plaque counts most 
strikingly. Some phages gave no plaques on 
media NaCl but gave high 
plaque counts when plated on the same me- 
dium without NaCl (with or without CaCl,), 
while other phages showed higher activity 
on NaCl than on CaCl, media. The expres- 
sion of phage activity is apparently influ- 
enced by a great many unknown factors. 
One of the phages lysed some strains with a 
medium containing NaCl and gave high 
plaque counts, but showed no activity 
against other strains on the same medium. 
These results suggest that a medium found 


containing 


optimal for one host-phage system might be 
quite unsuitable for another. Before a phage 
is typed against a large number of strains, 
an optimal medium must be developed. Bet- 
ter still, tests should be carried out with 
several different media, selected for their 
usefulness with particular strains. 


Serological Reactions 


Use of serological techniques, particularly 
those of agglutination and precipitation, has 


CHARACTERIZATION OF STREPTOMYCES SPECIES Ub 


been suggested for species identification of 
actinomycetes. Aoki (1935-1936) was thus 
able to differentiate between representatives 
of three genera, Actinomyces, Nocardia, and 
Streptomyces. By means of sonic vibrations, 
Ludwig and Hutchinson (1949) prepared an- 
tigen suspensions satisfactory for use in ag- 
glutinin and precipitin reactions and for the 
production of immune sera in rabbits. Use 
of such suspensions in the identification of 
actinomycetes was suggested by Yokoyama 
and Hata (1953) and Hata et al. (1953). A 
purified antigen of a streptomycin-producing 
strain was found active against immune sera 
of the same strain, but not against sera of 
other antibiotic-producing organisms. These 
investigators were thus able to establish the 
close relationship of luteomycin- and chlor- 
amphenicol-producing organisms. 

Ochoa and Hoyos (1953) found a correla- 
tion between microscopic morphology and 
serological reactions which made it possible 
to divide the actinomycetes into four 
groups: Group 1, including species of Ac- 
tinomyces and Nocardia; Group 2, made up 
largely of Nocardia; Groups 3 and 4, com- 
prising species of Streptomyces. Slack et al. 
(1951), however, found that antisera pre- 
pared with A. bovis brought about low titer 
agglutination of Nocardia and of two species 
of Streptomyces. They concluded that a close 
antigenic relationship exists between mem- 
bers of the genus Actinomyces and that there 
is a group relationship among Actinomyces, 
Nocardia, and Streptomyces. Okami (1956) 
found definite antigenic relationships be- 
tween strains of closely related forms of S. 
lavendulae. See also Tanaka et al., 1959. 


Chemical Composition 


A detailed study of the chemical composi- 
tion of cells of actinomycetes has been pre- 
sented in Volume I (pp. 158-163). 

The occurrence of specific chemical com- 
pounds in the cells of the organisms suggests 
possible differentiation between groups of 


actinomycetes. This is true, for example, of 
the occurrence of diaminopimelic acid, a 
constituent that may prove to be of generic 
rather than specific significance. Romano 
and Sohler (1956) and Sohler et al. (1958) 
have shown that cell walls of streptomycetes 
can be solubilized by lysozyme, suggesting 
the presence of a mucopolysaccharide; on 
the other hand, cell walls of nocardiae do 
not possess this property. 


Ecology 


The natural substrate of an organism, es- 
pecially diseased plants or animals, and com- 
posts of stable manures and plant residues 
at high temperatures, is of some systematic 
significance. Various attempts have been 
made to utilize the ecological characteristics 
of the actinomycetes as a basis of classifica- 
tion. Thus, the following rather broadly de- 
fined ecological categories have been pro- 
posed at various times to classify actino- 
mycetes: 

a. Animal parasites. 

b. Plant parasites. 

c. Soil inhabitants. 

d. Water inhabitants. 

e. Mesophilic forms. 

Thermophilic forms. 

Inhabitants of acidic (pH 3 to 6.5) sub- 

strates. 

h. Inhabitants of neutral to alkaline 
strates (pH 6.5 and above). 

The temperature at which an organism is 


oq 


sub- 


erown greatly affects the nature and amount 
of growth, the nature and extent of sporula- 
tion, and the degree of formation of soluble 
pigments. The optimum temperature for the 
growth of most species of Streptomyces is be- 
tween 25 and 30°C. Only a few of these or- 
ganisms are thermophilic. Abilities to grow 
under mesophilic and thermophilic condi- 
tions have been recognized as important 
criteria for establishing species and even 
genera of actinomycetes and other microor- 


ganisms. 


78 THE ACTINOMYCETES, Vol. II 


The optimum reaction for the growth of 
actinomycetes is pH 6.8 to 7.5. When these 
organisms are grown on complex organic 
media, and on many synthetic media, the 
reaction usually becomes alkaline. Some 
actinomycetes, however, are able to grow 
at pH 4.5 to 6.5 and even at pH 3.0 to 4.5. 
Such forms are not common, but the reaction 
of the substrate has been recognized as a 
potential diagnostic property. 

On the basis of their effects on dead resi- 
dues and upon living forms of life, actino- 
mycetes have been grouped as saprophytes 
and parasites, the latter being further 
grouped into plant and animal parasites. 
Thus we speak of ‘‘actinomycosis,” caused 
by A. bovis and A. 7sraelzi, and “nocardio- 
sis,’ caused by different species of Nocardia. 
We associate S. scabies with the ‘“seab”’ of 
potato tubers, and S. zpomoeae with a dis- 
ease of sweet potato roots. 


Genetics 


Little is known about the genetic proper- 
ties of actinomycetes and their possible 
bearing upon problems of classification. 
Certain observations have been made re- 
cently, however, which offer rather promis- 
ing leads in establishing species relationships. 
The concept of vegetative hybridization of 
Streptomyces cultures has been suggested. By 
repeated growth of a culture in a sterile fil- 
trate of sand-macerated mycelium of another 
culture, the former undergoes morphological 
and physiological changes. The significance 
of this phenomenon and its potential utiliza- 
tion for species characterization are still to 
be elucidated. Sermonti and Spada-Ser- 
monti (1956) demonstrated several types of 
recombination among ‘wild’? and mutant 
strains of S. 
violaceoruber). It has been brought out in 
Chapter 6 of Volume I that true hybrids can 
be obtained by mating two different mutant 


coelicolor (most probably S. 


strains of an actinomycete. Welsch (1958) 
suggested that mating may offer a conven- 


ient criterion for the practical definition of 
a species. The assumption was thereby made 
that a species is distinct if it does not cross, 
or gives only unfertile crosses with other 
similar species. 


Type Cultures 


An important, and often-used technique 
in species characterization of actinomycetes 
is that of comparing fresh isolates with 
type cultures. For the higher forms of plant 
life, species characterization is facilitated by 
study of preserved herbarium specimens. For 
microorganisms, special collections of named 
cultures are available for study. These cul- 
tures allow comparisons of living material, 
since dead or dried cultures are of but little 
significance. 

In establishing type cultures of actino- 
mycetes it Is important to keep in mind the 
fact that such cultures undergo considerable 
variation when grown for a long time upon 
artificial media. Some of the cultures may 
lose their ability to produce aerial mycelium 
and are thus deprived of properties of major 
diagnostic value. Unknown strains of Strep- 
tomyces free from aerial mycelium may even 
be considered as species of Nocardia. 

According to Pridham, * if reasonably fresh 
isolates were maintained on the proper media 
and preserved by lyophilization, the indi- 
vidual laboratory would experience far fewer 
difficulties than have been experienced in the 
past. Pridham reported that since 1953, with 
the use of these media and techniques, very 
rarely have strains been found that produce 
no aerial mycelium (which is generally well 
sporulated) on the isolation media; a very 
low incidence of strain degeneration has been 
noted in active cultures as determined by 
the methods of assessment, and all isolates 
have been routinely lyophilized. These 
lyophil tubes, opened from time to time, 
have been found to give cultures that are 


* Personal communication. 


CHARACTERIZATION OF STREPTOMYCES SPECIES 79 


Fiaure 21. Formation of straight sporophores by Streptomyces sp., X 1500 (Courtesy of Miss A. 
Dietz, Dept. of Microbiology, Upjohn Co., Kalamazoo, Mich.). 


equivalent to the original soil isolates, as 
determined by the methods of assessment 
used. 

The prior growth of the organism in soil 
media (sterile soil treated with a small 
amount of CaCO, , if acid, and with a half 
per cent of dried blood) or in carbon- or ni- 
trogen-poor media, its refrigeration or its 
lyophilization—each or all tend to prevent 


degeneration and thus preserve the original 
characteristics of the type culture. The cul- 
tures that have already degenerated will 
tend to regain their original properties as a 
result of such treatments. 

Shinobu (1958) suggested the following 
method for making a soil medium: Into a 
test tube of 1.5 em diameter place 7 g dried 
fertile soil; add 1.5 ml of 2 per cent solution 


80 THE ACTINOMYCETES, Vol. II 


of glycerol; make up the water content to 
about 20 to 25 per cent. Sterilize the tubes 
at 20 pounds for 20 minutes. The culture 
strains are inoculated on this medium and 
incubated at 28-30°C. When the growth of 
the organism is successful, white aerial my- 
celium appears first on the surface of the 
soil; when the culture matures the charac- 
teristic color of the aerial mycelium is pro- 
duced. 

There is always the danger that an old 
culture, transmitted from one laboratory to 
another, may either have become modified or 
have lost some of its original properties. It 
may have become contaminated, and the 
contaminant may eventually replace the 
original culture. One must also remember 
that different investigators have often based 
their descriptions of a particular species not 
upon the original culture but upon subse- 
quent isolates, which may or may not repre- 
sent the same species. 

Finally, many holo-type cultures are not 
available at all. Therefore, in some cases 
type cultures are not reported. When re- 
ported, they usually refer to the Institute of 
Microbiology Collection (IMRU), the 
American Type Culture Collection (ATCC), 
or to the Agricultural Research Service 
Culture Collection of the Northern Re- 
gional Research Laboratory, U.S. Depart- 
ment of Agriculture (NRRL). Other collec- 
tions include Kidige Technischen Hochschule 
(ETH) Zurich; Centraalbureau voor Schim- 
meleultures (CBS) Baarn; and Institute of 
Applied Microbiology, University of Tokyo 
(IAM). 


Standard Media 


Some media are more favorable than oth- 
ers for sporulation of Streptomyces cultures. 
In view of the importance of sporulation in 
characterizing a species (also in placing an 
organism in the proper genus), it is essential 
to select favorable media. Furthermore, since 
some forms tend to lose the property of 


sporulation on continued growth, special 
precautions must be taken in preserving 
such cultures. The loss of aerial mycelium 
may be reversible or irreversible. Since non- 
sporulating streptomycetes may resemble 
nocardiae and since certain nocardiae have 
been reported to produce aerial mycelium 
and spores similar to those of typical Strep- 
tomyces cultures, the element of confusion 
between the two genera always exists. 


Description of Streptomyces Species 


It is commonly believed that to charac- 
terize a species it 1s essential to describe a 
large number of its morphological and phys- 
iological properties. This procedure is not 
always helpful, especially if based upon un- 
reliable criteria. The medium may not be 
readily duplicated, or conditions of growth 
may be different, or the inoculum may not 
be prepared in the same way. Because of 
these and other variations, many cultures 
recently isolated have been described as new 
species. Another reason is that it is much 
vasier to create a new species than to at- 
tempt to correlate the characteristics of a 
freshly isolated culture with those of known 
species already described in the literature. 
Numerous species also have been 
created to facilitate the obtaining of patents. 

Hesseltine ef al. (1954) suggested that the 
following steps be taken in the taxonomic 


hew 


study of a Streptomyces species: 
1. Collection of strains on 
pigmentation of aerial mycelium. 
2. Study of the morphology of strains 


the basis of 


erowlng on a number of media favorable to 
sporulation. 

3. Examination of the color of spores of 
strains growing on optimum sporulating me- 
dia. Five color groups were recognized: (a) 
lavender, red, or pink; (b) blue, blue-green, 
or green; (c) yellow; (d) white; (e) gray, 
gray-brown, olive-gray, or dark gray. 

4. Study of cultural characters of strains 
on various synthetic and organic media. 


CHARACTERIZATION OF 


5. Analysis of certain physiological and 
biochemical properties, notably action on 
gelatin, starch, milk, and peptone-iron agar; 
nitrate reduction; utilization of carbon and 
nitrogen compounds; antibiotic action, com- 
prising formation of and sensitivity to anti- 
bioties. 

6. Identification 
known species, and preservation of cultures. 

Pridham* emphasized that recently he has 
been placing principal emphasis on micro- 


of new strains with 


morphology, secondary emphasis on chro- 
mogenicity (deep brown to black diffusible 
pigments), and tertiary emphasis on color of 
aerial mycelium. 

In an effort to determine whether freshly 
isolated cultures can be identified on the 
basis of published descriptions and what con- 
ditions justify the creation of new species, 


several obvious comparisons were made 
(Waksman, 1957). Certain strains that 


might be included in various important 
species or group-species were critically ex- 
amined. The following conclusions were 
reached: 


At present, various morphological, cultural, 
and biochemical properties are known which make 
it possible to establish definitely certain distinct 
species among the actinomycetes. Some of these 
characters are constant within certain conditions 
of nutrition and environment, others are varia- 
ble. Certain additional properties may be re- 
quired in order to establish the degree of varia- 
tion of a culture before it can be recognized as a 
new species. 

Certain categories of relationships among the 
actinomycetes must be taken into consideration 
in order to establish definitely the systematic 
position of a given culture. These may be briefly 
summarized as follows: 

1. On the basis of all the accumulated evi- 
dence, actinomycetes are shown to belong defi- 
nitely to the bacteria. 

2. The position of the true actinomycetes in 
relation to related bacterial forms, notably the 


* Personal communication. 


STREPTOMYCES SPECIES SI 
mycobacteria and corynebacteria, must be recog- 
nized; this is true especially of certain nocardial 
types. 

3. The generic interrelationships among the 
actinomycetes are highly significant. The separa- 
tion of members of the genus Streptomyces from 
those of Nocardia is difficult, especially when one 
is dealing with nonsporulating forms of the first 
and sporulating forms of the second. The recent 
addition of two new genera, Actinoplanes and 
Streptosporangium, and the recognition of certain 
thermophilic groups as separate genera add fur- 
ther problems to these generic interrelationships. 

4. Within each genus, certain groups, species- 
groups, or series must be recognized. A combina- 
tion of morphological and cultural 
permits the establishment 
Some of these comprise a large number of forms 
with many variable characteristics. 


properties 


of species-groups. 


5. Differentiation of individual species within 
each group is based upon a combination of cul- 
tural and biochemical properties. The production 
of specific antibiotics and the utilization of differ- 
ent sugars are ample illustrations of this. 

6. Cognizance of the strains and varieties 
within each species must finally be taken. This 
may be based upon certain qualitative properties, 
such as sensitivity to phages, or quantitative 
properties, such as production of a given anti- 
biotic, vitamin, or enzyme, or sensitivity to a 
given antibiotic. 


The fact that a culture becomes important 
for the production of a particular metabolic 
product, such as an antibiotic, an enzyme, 
or a vitamin, may impart to the culture 
particular significance for characterization 
purposes. 

The existence of physiologic races or varie- 
ties among species of actinomycetes, espe- 
cially among those placed in the genus 
Streptomyces, has been fully recognized. 
Just as in improving higher forms of life 
one is always faced with the selection of 
varieties resistant to disease, or giving higher 
yields, or having other desirable qualities, 
so one must select strains of actinomycetes 
on the basis of resistance to phage or of pro- 
duction of higher yields of a given antibiotic 
or other metabolic product. 


Ghva ip tier 49 


Systems of Classification and 
Identification of Groups and 
Species of the Genus 
Streptomyces 


Principles of Separation of Genera 


The historical background and various 
systems of classification of the order Actino- 
mycetales in general and of the actinomy- 
cetes in particular have been discussed in 
detail in Chapter 4 of Volume I. The princi- 
ples underlying the generic and specific 
separation of the organisms are presented 
in Chapter 4 of the present volume. The 
variability and overlapping among genera 
and species have been emphasized in Chap- 
ter 6 of Volume I. Certain important factors 
pertaining specifically to the genus Strepto- 
myces must be considered before any dis- 
cussion is presented of the separation of this 
genus into subgenera, series (sSpecies-groups), 
species, and varieties. 

Among the factors that must be empha- 
sized in any attempt to classify actinomy- 
cetes, the following three are most impor- 
tant: (a) the nature of the substrate (or 
vegetative) growth and the nature of the 
aerial mycelium, if any; (b) the degree of 
variability of the cultures; and (c) the effect 
of the composition of the medium. To facili- 
tate recognition of the organisms and to 
establish constant and variable differences 
fol purposes, well-defined 
media and standard conditions of cultiva- 
tion must be used. 


classification 


Actinomycetes are differentiated from the 
true bacteria by their filamentous growth 
and by their true branching. It is often 
dificult, if not impossible, to distinguish 
between the profuse branching of certain 
mycobacteria and the short-lived mycelium 
of the nocardias, except for the fact that 
the latter produce mycelium consistently 
in the early stages of their development. 
There is a gradual transition between the 
mycobacteria and the nocardias. It also is 
often difficult to differentiate between the 
nocardias and the streptomyces. The latter 
are characterized the constant and 
marked nature of their aerial mycelium, 
whereas 


by 


characterized 
largely by the transitory and undifferenti- 
ated nature of this mycelium. 


the nocardias are 


In establishing differences between no- 
cardias and streptomyces, one must con- 
sider the following factors: 

1. Nocardias usually have been consid- 
ered incapable of forming aerial mycelum 
that could be differentiated from the sub- 
strate mycelium. It also has usually been 
assumed that no spirals are ever formed 
the mycelium. 
and Mihm 


that certain nocardias are able to form aerial 


from Recently, however, 


(Gordon (1958) have reported 


mycelium similar to that of streptomyces 


GROUPS AND SPECIES OF 


and that spirals also may be formed. A 
streptomycete forms a characteristic aerial 
mycelium. This property may be lost, how- 
ever, on continued cultivation or under 
special conditions of treatment. The aerial 
mycelium frequently develops characteristic 
spirals, tufts (Fig. 22), or verticils (whorls). 

2. A streptomycete usually multiples by 
the concentration and fragmentation of the 
protoplasm within a filamentous cell, fol- 
lowed by the dissolution of the cell mem- 
brane. The fragmented portions of the my- 
celium usually develop, under favorable 
conditions, into fresh mycelium, either by 
germ tubes or by lateral budding. Spores or 
conidia are produced. The substrate my- 
celium does not segment spontaneously into 
bacillary or coccoid forms, but remains non- 
septate and coherent even in old cultures, 
thus forming the characteristic tough tex- 
tured, leathery growth. 

3. In nocardias, the aerial hyphae are 
believed to represent an upward extension 
of the substrate mycelium, and usually do 
not exhibit any differentiated protoplasm. 
When a streptomycete loses its capacity to 
produce aerial hyphae, a form analogous to 
that of a nocardia may result, except for 
the structure of the mycelium that 
faculty of the degenerated culture to regain 


and 


the lost capacity. 

4. Another difference between nocardias 
and streptomycetes is the acid-fastness or 
partial acid-fastness of some of the forme! 
when grown in certain media; the latter are 
never acid-fast. 

As pointed out in Chapter 1, Gordon and 
Smith (1955) proposed six distinctive char- 
acters for the separation of the two genera. 
These criteria are: (a) colony structure; (b) 
casein hydrolysis; (¢) dissolution of tyrosine 
and xanthine; (d) acid production from glu- 
cose and glycerol; (e) lack of acid formation 
from arabinose, xylose, lactose, mannitol, 
and inositol; and (f) utilization of acetate, 
propionate, pyruvate, malate, and succinate. 

Strains of organisms giving positive re- 


GENUS STREPTOMYCES 835 


Ficure 22. Tuft formation in aerial mycelium 
of the Streptomyces griseus type. 


sults in five or six of the physiological tests 
belong to Streptomyces; strains with four 
to six negative reactions, to Nocardia. 
Although considered as somewhat arbi- 
trary, these criteria allowed clear-cut generic 


separation of 97 per cent of 251. strains 
studied, regardless of their morphological 


variation. Ninety-six per cent of the strains 
received as Streptomyces were positive in 
five or six of the following reactions: hy- 
drolysis of casein, dissolution of tyrosine, 
and acid production from xylose, mannose, 
maltose, and lactose. Strains that no longer 
formed aerial hyphae and spores, but known 
ot 
ones, also were positive in five or six of 
the 


beled Nocardia gave negative results in four 


to be descendants typical sporulating 


these tests. Two-thirds of strains la- 
to six of the same tests. Of the remaining 
third of the straims 
24 had the 


strains of Streptomyces and were assumed to 


received Nocardia, 


as 


same reactions as accepted 


84 THE ACTINOMYCETES, Vol. II 


be mislabeled; seven were listed temporarily 
as Intermediates between the two genera. 

The differentiation between Streptomyces 
and the other genera of actinomycetes is 
not very difficult. The formation of aerial 
mycelium and the manner of sporulation 
are markedly distinct for Streptomyces as 
compared to A/icromonospora. Species of 
Thermoactinomyces also produce an aerial 
mycelium, similar to that of species of 
Streptomyces, but they form single spores, 
similar to those of A/icromonospora. The 
other thermophilic genera, as well as the 
genera Waksmania (Microbispora*), Actino- 
planes, and Streptosporangium also can be 
differentiated from Streptomyces, as shown 
in Chapters 8 to 11. 

Among the numerous species belonging 
to the various genera of actinomycetes, 
those of the genus Streptomyces are by fai 
the most important, largely because of their 
wide distribution, their greater abundance, 
and their ability to produce antibiotics and 
vitamins and to carry out important chemi- 
cal conversions. Hence a detailed considera- 
tion of this genus is justified. 


Description of Genus Streptomyces 


Streptomyces species produce a_ well-de- 
veloped mycelium. The diameter of the 
hyphae seldom exceeds 1.0 u and is usually 
only 0.7 to 0.8 un. The hyphae vary greatly 
in length: some are long with limited branch- 
ing; others are short and much branched. 
The substrate mycelium does not form cross 
walls; it does not break up into rod-shaped 
and coccus-like bodies. Reproduction occurs 
by means of spores or by bits of mycelium. 
Spores or conidia are formed in_ special 
spore-bearing hyphae or sporophores which 
arise from the aerial mycelium either mono- 
podially or in the form of tufts or verticils. 

* Both designations were published, in different 


journals, the same month and the same year. 


Priority has not been definitely established. 


The sporulating hyphae are straight or 
curved. The curvatures range from mere 
waviness to perfect spirals, which may be 
compact, in the form of fists, or long and 
open (Fig. 23). 

The spores of streptomycetes comprise 
four types: smooth, warty, spiny, or hairy. 
About one-third of the gray- to brownish- 
spored species were found (Tresner et al., 
1960) to form spiny, warty, or hairy spores; 
the remainder were smooth-spored. All the 
blue- to blue-green-spored forms had spiny 
spores. White, vellow, cream, or buff types 
had smooth-walled spores. All the pinkish- 
tan-spored group had smooth spores, with 
the exception of S. erythreus and S. pur- 
purascens Which had spiny spores. The con- 
clusion was also reached that, because of 
the variation of spore size and shape, those 
properties are of lmited usefulness for 
taxonomic differentiation. 

The growth of Streptomyces ‘‘colomies’ 
on artificial media is smooth or lichenoid, 
hard and densely textured, raised, and ad- 
hering to the medium. The colony is usually 
covered completely or partially (in the form 
of spots or concentric rings) by aerial my- 


? 


celium, which may be variously pigmented, 
depending on the species and on the com- 
position of medium. In liquid media, es- 
pecially in shaken cultures, growth of strep- 
tomyces is usually in the form of flakes, 
which gradually fill the container, or in the 
form of spherical growths; the former type 
of growth is the more desirable from the 
point of view of antibiotic production. 

Many of the cultures, either in the form 
of colonies on the surface of solid media or 
as flaky growth in submerged culture, may 
undergo rapid lysis. The production of anti- 
biotics usually corresponds with the lysis 
of the cultures. Frequently, the lysis is 
brought about by a phage, known as actino- 
phage, which exerts an injurious or destruc- 
tive effect upon the mycelium. 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 85 


FIGURE 23. 


3ull. Torrey Botan. Club 82: 111, 1955). 


Classification Systems of the Genus 


Streptomyces 


Early Systems of Classification 


Although the earlier systems of classifica- 
tion of actinomycetes were also supposed to 
be concerned with all the forms usually in- 


cluded in this group, they represented 


Various strains of Streplomyces californicus (Reproduced from: 


Jurkholder, P. 


Re el al. 


largely those forms that are now included 
in the genus Streptomyces. This is true, for 
example, of the first classification of San- 
felice (1896), and the subsequent ones of 
Krainsky (1914) Waksman 
Curtis (1916). Only the more comprehensive 


and of and 


and more recent systems are presented 


here. The earlier ones were given in Chapter 


t of Volume L. 


86 THE ACTINOMYCETES, Vol. II 


1. WAKSMAN AND CURTIS (1916) SYSTEM 


This system was based upon formation of 
soluble pigments in organic media, rate of 
gelatin liquefaction, and structure of aerial 
mycelium. 

A. Gelatin rapidly liquefied; no brown pigment. 
I. Spirals formed. 


1. No soluble pigment on synthetic media. 
Actinomyces Rutgersensis 


2. Pigment formed on synthetic media. 
a. Pigment dark blue. 
Actinomyces violaceus-Caesert 
b. Pigment brown. 
Actinomyces diastaticus 
II. No spirals. 
1. No soluble pigment. 
a. Growth orange-red, aerial mycelium 
white. 
Actinomyces albosporeus 


Fraure 24. Sporophores of Streptomyces sp. producing spirals, X 1500 (Courtesy of Miss A. Dietz, 
Dept. of Microbiology, Upjohn Co., Kalamazoo, Mich.). 


———— 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 87 


b. Growth rose-colored, aerial mycelium 
rosy. 
Actinomyces Fradit 
ce. Growth a mixture of white and yel- 
low. 
al. No conidia. 
Actinomyces albo-flavus 
bt. Abundant conidia. 
a2. Conidia rod-shaped, powdery, 
gray-yellow. 
Actinomyces griseus 
b?. Conidia spherical and oval, 
growth compact, citron-yel- 
low. 
Actinomyces citreus 
d. Growth at first colorless, then brown 
to black. 
al. Aerial mycelium white. 
Actinomyces alboatrus 
bt. Aerial mycelium dark gray. 
Actinomyces Lipmanit 
. Soluble pigment produced. 
a. Soluble pigment green. 


bo 


Actinomyces Verne 
b. Soluble pigment dark blue. 
Actinomyces violaceus-niger 
B. Gelatin liquefied; brown pigment 
formed. 
I. Spirals produced. 
a. Growth rose-colored; aerial mycelium 
rosy. 


rapidly 


Actinomyces roseus 
b. Growth colorless; aerial mycelium 
golden brown. 
Actinomyces aureus 
ce. Growth slightly brown; aerial mycel- 
ium white. 
Actinomyces Halstedii 
Il. No spirals. 
1. No soluble pigment on synthetic media. 
a. Growth red to red-orange; no aerial 
mycelium. 
Actinomyces Bobili 
b. Growth white; aerial mycelium white. 
al. Aerial mycelium thin, rare, net- 
like. 
Actinomyces reticult 
b!. Aerial mycelium thick, white to 
gray. 
Actinomyces albus 
2. Soluble brown pigment produced on 
synthetic media. 
a. Aerial mycelium white, abundant. 
Actinomyces diastato-chromo- 
genus 


b. Aerial mycelium white, 


late or not at all. 


produced 


Actinomyces 
group 
ce. Growth green; aerial mycelium white. 


chromogenus 


Actinomyces — virido-chromo- 
genus 
C. Gelatin slowly liquefied; no soluble pigment. 
I. Spirals produced in aerial mycelium. 
1. Soluble red and blue pigments. 
Actinomyces violaceus-ruber 
2. No soluble pigment; substrate growth 
red. 
Actinomyces Californicus 
Il. No spirals produced in aerial mycelium. 
1. Growth yellow; no soluble pigment. 
Actinomyces parvus 
2. Growth tends to crack; soluble brown 
pigment. 
Actinomyces exfoliatus 
D. Gelatin slowly hquefied; brown pigment pro- 


duced. 
I. Spirals produced; aerial mycelium laven- 
der. 


Actinomyces lavendulae 
II. No spirals. 
1. Growth yellow; aerial mycelium gray. 
Actinomyces flavus 
2. Growth colorless; aerial mycelium 
purplish-white. 
Actinomyces purpurogenus 
3. Growth black; aerial mycelium scant. 
Actinomyces — erithrochromo- 
genus 
4. Growth purple; no aerial mycelium. 
Actinomyces purpeo-chromo- 


Genus 
2. WAKSMAN SYSTEM (1919) 


This was a modification of the previous 
system and was based upon a study of 41 
species. An examination was made of the 
morphology of the aerial mycelium on two 
media; growth, aerial mycelium, and solu- 
ble pigment on 12 different media; various 
biochemical properties, such as carbon and 
nitrogen utilization, proteolytic activities, 
diastase and invertase formation, reduction 
of nitrate to nitrite, and change in reaction 
of medium. A brief outline is presented here: 
A. Soluble pigment produced on organic 

media. 


Py 
ia 
4 
{/ 
(/ 


P qi a 
- <Gi frre 7re se e 


eee 
ae, fey 
CLIT 


a jm yaya! 


v. = 


14 
CLrechs/2° 
ya gel 
FIGURE 25. One of the early studies on the strueture of the aerial mycelium and the manner of sporu- 
lation of Streptomyces cultures (Reproduced from: Drechsler, C. Botan. Gaz. 67: 66-83, 147-168, 1919). 


SS 


GROUPS AND SPECIES OF 


I. Pigment deep brown. 
Il. Pigment faint brown, golden yellow, 
or blue. 
B. No soluble pigment on organic media. 
I. Strongly proteolytic. 
Il. Weakly proteolytic. 

The need for the recognition of species- 
groups was also emphasized: ‘‘All the cul- 
tures should be divided into groups, the 
representatives of which have 
morphological, physiological and cultural 
characters. These species-groups may show 
slight variations within the groups, when 


common 


several representatives are compared, but 
all of them possess in common the main 
distinguishing characters of the species, and 


are distinctly different from any other 
species-group.”’ 
3. JENSEN’S SYSTEM (1930) 

Jensen modified the above system of 


Waksman, in describing 90 strains of actino- 
mycetes, largely streptomycetes, isolated by 
him. These strains were divided into several 
species-groups. 


A. No pigment produced on protein media. 
1. Red or blue pigments in synthetic media; 
marked reduction of nitrates. 
Actinomyces violaceus-ruber 


to 


No red or blue pigments. 

a. Typical golden pigment in all synthetic 
media. 

Actinomyces fulvissimus 

b. Pigment not typical; abundant aerial 

mycelium. 

al. Aerial mycelium on synthetic media 
dark slate-gray; sulfur- 
yellow pigments sometimes formed. 
a2. Vegetative mycelium on synthetic 


lemon- or 


agar light colored. 
Actinomyces cellulosae 
b?. Vegetative mycelium on synthetic 
agar turning dark. 
Actinomyces olivaceus 
. Aerial mycelium greenish- or yellow- 
ish-gray; very rapid liquefaction of 
gelatin and blood-serum. 
a®. Aerial mycelium greenish. 
Actinomyces griseus 


GENUS STREPTOMYCES 89 
b?. Aerial mycelium yellowish. 
Actinomyces griseoflavus 
B. Typical brown pigment in protein media 
(“chromogenus’’ species). 
1. Deep brown growth and pigment in all syn- 
thetic media. 
Actinomyces phaeochromo- 
genus 
2. Pigment in synthetic media of other color or 
absent. 
a. Aerial mycelium absent or in 
typical red vegetative mycelium. 


traces; 


Actinomyces bobili 
b. Aerial mycelium more or less abundant. 
al. Typical 
agar. 


red pigment in synthetic 


Actinomyces — erythrochromo- 
genus 
. Pigment not red. 
a®, Aerial mycelium rose to cinna- 
mon-brown. 
Actinomyces roseus 
b?. Aerial mycelium abundant, char- 
acteristic lead-gray; hght brown 
pigment in synthetic media. 
Actinomyces diastatochromo- 
genus 


Jensen 
term 


contribution, 
the 
actinomycetes 


subsequent 
(1931) emphasized again that 
“species” applied — to 
should be used in the sense of Waksman’s 
“species-groups,” ora “broad group of strains 
in 


In a 


as 


agreeing celtain outstanding morpho- 
logical and biological features’; otherwise, 
ce . . . on . 
every strain of actinomycetes isolated from 
a plating from an ordinary soil could then 
be raised to the rank of species.” 


4. KRASSILNIKOV SYSTEM (1941) 


This system was based primarily upon 
the morphology of sporophores of the cul- 
tures and the shape of their spores, and 
secondarily upon the pigmentation of the 
cultures. 

A. Sporophores branching monopodially. 
I. Spiral-shaped sporophores, produced on 
hyphae of aerial mycelium. 
1. Spores spherical or oval. 
a. Cultures colorless, not producing any 
pigmentation. 


90 


b. 


Cc. 


d. 


e. 


THE ACTINOMYCETES, 


al. Aerial mycelium white. 
a®, Saprophytes, hving on dead 
material. 

Actinomyces albus 

b2. Parasites, living on plants. 

Actinomyces totschidlowskii 
bt. Aerial mycelium dark gray. 

Actinomyces griseus 
cl, Aerial mycelium green. 

Actinomyces glaucus 
Cultures pigmented blue. 

a'. Pigment of the anthocyanin type, 
similar to litmus. 

Actinomyces coelicolor 
bt. Blue pigment not changing with 

acidity of medium. 

Actinomyces cyaneus 
Cultures violet, forming two basic 
pigments (red and blue), both dis- 
solved into the substrate. 
al. Cultures not forming any fluores- 

cent substance in liquid media. 

Actinomyces violaceus 
b!. Cultures producing in synthetic 

media a fluorescent substance of 
blue-green color similar to pyo- 
eyanin. 

Actinomyces pluricolor 
Cultures black-violet, forming red 
and blue pigments, as well as a brown 
pigment of the type of melanin, which 
changes the violet color of the culture 
to violet-black. 

Actinomyces violaceus-niger 
Cultures red-colored, producing pig- 
ments insoluble in water, of the hpo- 
actinochrome type; color of medium 
not changing with acidity. 
al. Cultures not forming any brown 

or black pigments; they are al- 
sometimes with a 
brownish tinge, but not black. 


ways red, 


Actinomyces ruber 
bt. Cultures producing on synthetic 
media, in addition to pigments, a 
black or dark brown substance 
which gives the culture a red- 
brown to black color. 
Actinomyces melanocyclus 
Cultures yellow, 
brownish-yellow. 


citron-yellow, or 


al. Saprophytes. 
Actinomyces flavus 

b!. Living on plants. 
Actinomyces setonii 


i 


lave 


Vol. II 


Cultures orange. 
al, Saprophytes. 
Actinomyces aurantiacus 
b!. Parasites. 
Actinomyces phenotolerans 
Cultures green or brownish-green. 
Actinomyces viridochromo- 
genes 
Cultures black, producing a pigment 
of the melanin type. 
Actinomyces niger 
Cultures pigmented dark brown, but 
not black. 
al, Saprophytes. 
Actinomyces chromogenes 
b!. Plant parasites. 
Actinomyces gracilis 


2. Spores cylindrical or elongated. 


a. 


loys 


— 


al 


Cultures colorless. 
Actinomyces longisporus 

Cultures red, mostly 

straight. 

al. Saprophytes. 
Actinomyces 


sporophores 


longis porus - 
ruber 
b!. Parasites. 
a2. Living in bodies of men and 
animals. 
Actinomyces spumalis 
2. Living on plants. 
Actinomyces salmonicolor 


Y 
Ss 


Cultures orange. 
Actinomyces fradiae 


. Cultures yellow. 


al. Saprophytes. 
Actinomyces 
flavus 

b!. Parasites living on plants. 


longisporus- 


Actinomyces scabies 
Cultures citron-yellow. 

Actinomyces virgatus 
Cultures green. 

Actinomyces viridans 
Cultures brown or chocolate-colored. 
Actinomyces halstedit 

Cultures black. 
Actinomyces nigrificans 


Sporophores straight or wavy, but not 


spiral. 
1. Spores produced by means of fragmenta- 


tion of plasma within cells. 


a. 


Spores spherical or oval. 
al. Cultures colorless. 
Actinomyces globisporus 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 91 


b!. Cultures green. 
a®. Saprophytes. 
Actinomyces viridis 
b2. Plant parasites. 
Actinomyces cretaceus 
e!. Cultures brown. 
Actinomyces globosus 
b. Spores cylindrical or elongated. 
alt. Cultures colorless. 
Actinomyces candidus 
b'. Cultures pigmented. 
Actinomyces cylindrosporus 
2. Spores produced by means of segmenta- 
tion of aerial hyphae. 
a. Cultures colorless. 
Actinomyces farinosus 
b. Cultures pigmented red. 
Actinomyces oidiosporus 
Actinomyces rectus 


~ 


. Cultures vellow-orange. 
Actinomyces longissimus 
d. Cultures blue. 
Actinomyces caeruleus 
. Cultures brown. 
Actinomyces fumosus 


a) 


B. Sporophores produced in verticils. 
I. Sporophores straight. 
Actinomyces verticillatus 
Il. Sporophores spiral-shaped. 
L. Spores spherical, oval. 
Actinomyces reticuli 
Actinomyces reticulus-ruber 
2. Spores cylindrical, elongated. 
Actinomyces circulatus 


Detailed consideration was given to some 
of the larger groups of the genus, notably 
to the Albus and Flavus groups, as will be 
shown in Chapter 6. 


5. WAKSMAN AND HENRICI SYSTEM (1943) 


This system was used in the last two edi- 
tions of Bergey’s Manual of Determinative 
Bacteriology (1948, 1957). It may be listed 
here among the earlier was 
based primarily upon the ecology of the 


systems. It 


organisms, production of soluble pigments 
in organic and synthetic media, and proteo- 
lytic properties. 
A. Saprophytes; psychophilic to mesophilic. 
1. Soluble pigment on organic media 
other than brown, or faint brown. 


a. Pigment absent or faint brown 
only. 

b. Pigment blue. 

c. Pigment at first 


brown, ete. 


green, becoming 


2. Soluble pigment on organic media 
brown. 
3. No soluble pigment produced in or- 


ganic media. 
a. Proteolytic action strong. 
b. Proteolytic action limited. 
c. Proteolytic action very weak. 
B. Saprophytes; thermophilic. 
1. Yellowish 
static. 
2. Dark-colored 


potato; nondiastatic. 


growth on potato; dia- 


abundant growth = on 
5}. Thermotolerant cultures. 

C. Plant parasites or cultures isolated from 
diseased plants or from soil in which 
diseased plants were grown. 

1. Isolated from potato scab or from 
soil in which scabby potatoes were 
erown. 


bo 


Grown on or isolated from sweet 


potatoes. 


3. Isolated from scab on mangels and 
sugar beets. 

D. Isolates the 
animal body, hyphae often show clavate 


from animal tissues; in 
enlargements at the ends. 
1. Limited proteolytic action. 


2. Strong proteolytic action. 


— 
[ter 


Produce only substrate growth and no 
aerial mycelium. 
The last system, like the earlier ones, may 
now be considered as of purely historic in- 
terest. For the purpose of this treatise, it 
has been considerably modified. It has been 
greatly enlarged to include all newly de- 
scribed organisms belonging to the genus 
Streptomyces. Some of the features of the 
older system, notably those pertaining to 
ecology, have been left out altogether. 


92 THE ACTINOMYCETES, Vol. II 


Recent Systems of Classification 


Several systems for classifying species 
belonging to the genus Streptomyces have 
recently been proposed. Some of these have 
been selected for detailed examination. They 
are based largely upon morphology, cultural 
and biochemical properties, or combina- 
tions of these. 


1. HESSELTINE, AND PRIDHAM 


SYSTEM (1954) 


BENEDICT, 


In this system, emphasis was laid upon 
morphology as the basis for separation of 
the genus into five basic groups. After a 
study of hundreds of cultures on a variety 
of media, the conclusion was reached that 
the morphology of any one strain of Strepto- 
myces is essentially the same on any medium 
where sporulation occurs. The major groups 
were subdivided into a number of subgroups 
on the basis of cultural properties, pigmen- 
tation of spores, and other criteria. 

I. Sporophores not restricted in length, 
bearing fertile branches in verticils, 
with spores more or less strongly at- 
tached. 

1. Fertile branches in simple verticils, 
branches not ending in spirals. 
Fertile branches in simple verticils, 
branches ending in spirals. 

3. Fertile branches with compound 
verticils, branches not ending in 


bo 


spirals. 

II. Sporophores with branches all straight, 
never ending in spirals; verticils ab- 
sent. 

III. Sporophores predominantly in tufts, 
never verticillate; outline of branches 
flexuous and irregular. 

IV. Sporophores with branches ending in 

absent; sporo- 

stalks bearing 
as short stalks 


spirals, verticils being 

phores either as long 

very short branches, or 

bearing branches irregularly. 

1. Branches ending in open spirals with 
many turns. 


2. Branches ending in closed spirals 
with few turns, thus appearing as 
tight knots. 

V. Sporophores with long and_ straight 
branches with spirals of large diameter 
at their ends; spirals usually with only 
a few turns, never verticillate. 

No strains were observed in which the 
sporophores were unbranched, except when 
they were growing under unfavorable con- 
ditions or where degenerated type cultures 
were studied. 

Seven major Streptomyces groups were 
thus created as indicated by the following 
key: 


I. Sporophores produce verticils; spores not 
readily separating; aerial mycelium white, 
pink, lavender, or tan. 

Group |. Streptomyces reticuli 
1. Verticil branches simple. 
a. Sporophores straight. 
b. Sporophores spiral-shaped. 
2. Verticil branches compound. 
a. Ultimate branches straight. 
b. Ultimate branches spiraled. 

II. Sporophores not producing any  verticils; 
spores readily separate; color of aerial myce- 
lium often not pink, white, lavender, or tan. 
1. Spirals always formed; color of aerial my- 

celium blue, blue-green, or green. 
Group II. Streptomyces viridochro- 
mogenes 
2. Spirals may or may not be formed; color of 
aerial mycelium different. 

a. Spirals never formed, tufts often present ; 
color of aerial mycelium greenish-tan or 
tan, never white. 

Group III. Streptomyces griseus 
b. Spirals produced; color of aerial myce- 
lium lavender, red, pink, or nearly tan. 
Group IV. Streptomyces lavendulae 
Sporophores straight. 
a. Spores white or nearly so. 


vw 


Group V. Streptomyces albus 
b. Aerial mycelium never white. 
al. Aerial mycelium yellow. 
Group VI. Streptomyces parvus 
bt. Aerial mycelium gray, gray-brown, 
olive-gray, blackish-gray. 
Group VII. Gray-spored group. 


It was suggested that the last group could 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 93 


be subdivided on the basis of spiral forma- 
tion. 


2. FLAIG AND KUTZNER SYSTEM (1954, 1960) 

Flaig and Kutzner (1954) and Kutzner 
(1956) studied about 2000 Streptomyces cul- 
tures, 63 of which were authentic species 
and the rest fresh soil isolates. They charac- 
terized their cultures by several criteria in- 
cluding cultural characteristics on complex 
and synthetic media, morphology of the 
aerial mycelium, physiological properties, 
and antibiotic activity against five test or- 
ganisms. On the basis of these studies, a 
key was prepared. At first the material was 
divided into ‘“‘groups’? on the basis of the 
color of the aerial mycelium on oatmeal 
agar, six spore colors being recognized. The 
gray color group was further divided, on the 
basis of the color of the substrate mycelium 
and soluble pigment on this medium, thus 
resulting in the 10 groups shown in Table 9. 

Later, however, Flaig and Kutzner (1960) 
reached the conclusion that the subdivision 
of the gray-spored color group may lead to 
difficulties when new isolates have to be 
placed in one of these groups, although this 
system proved to be quite useful in studying 
many strains at the same time. 

Each group was further divided into sub- 
groups (altogether 382) on the basis of pig- 


ment formation on glucose-peptone agar, 
morphology of the aerial mycelium, anti- 
biotic activity against five test organisms, 
and cultural several 
media. The shape of the spores (observed 
with electron microscope) was given for 175 
of the 382 subgroups. Various authentic 


characteristics on 


species were included in these subgroups 
according to their characteristic properties. 
The following species were placed in the 
various groups. 

Group I: S. albus, S. griseus, S. chryso- 
mallus, S. coelicolor, S. cali- 
fornicus 

Group II: — S. longispororuber, S. bobiliae, 
S. roseochromogenes, S. vene- 
zuelae, S. phaeochromogenes 
Group III: 8S. lavendulae, S. xanthophaeus 


Group IV:  S. flavogriseus, S. globisporus, 
S. flaveolus 
Group V: S. diastaticus, S. globosus 


Group VI:  S. flavus 
Group VII: S. craterifer, S. griseolus, S. 
halstedii, S. hygroscopicus, S. 
aureofaciens, S. violaceoruber 
Group VIII: S. purpurascens 
Group IX: S. 
genes, S. chartreusis 
Group X:_ iS. 
prasinopilosus 


cyaneus, S. viridochromo- 


hirsutus, S. prasinus, S. 


TABLE 9 


Grouping of the genus Streptomyces according to characteristic coloration (Flaig and Kutzner, 1960) 


Group Aerial mycelium Substrate mycelium and soluble pigment 
I Yellowish to yellow-gray Colorless, brownish, reddish, greenish 
II Light rose to reddish Colorless, orange, greenish, brownish to 
g 
dark brown, pink to dark red 
IIL Gray-rose (lavender) Colorless, orange, brownish to dark brown 
i io 
IV Light gray to gray Yellowish-green to green 
. aS . S S 
V Gray Brown 
VI White to gray (cottony) Colorless, brownish to brown 
VII Gray (dusty) Colorless, greenish-gray, brownish-gray, 
2 : g era) Bra) 
orange-brownish, red or blue violet 
VIII Light gray or pink Violet 
IDS Blue Blue-purple, bluish-green, brownish 
pur} 
x Green Colorless, greenish, brownish to rose-red 


94 THE ACTINOMYCETES, Vol. II 


3. SYSTEM OF YAMAGUCHI AND SABURI (1955) 


Yamaguchi and Saburi also used morpho- 
logical features as the primary basis for the 
separation of the genus Streptomyces into 
groups, and physiological characteristics 
for further separation. Although they were 
concerned primarily with species of Strepto- 
myces possessing antitrichomonal properties, 
their system may apply to the genus as a 
whole. 


I. Sporophores straight, tuft-forming tendency 
in the margin; no verticils or spirals. 
1. Aerial mycelium gray. 

a. Soluble pigment on protein media. 
Light purple, reddish-purple, purplish- 
brown, sometimes yellowish-brown. 

Streptomyces purpeofuscus 

b. No soluble brown pigment. 

Streptomyces fasciculus 

Aerial mycelium pale yellowish-green. 

a. Soluble pigment on protein media 
brown—ATCC Culture No. 3309. 

b. Soluble pigment brown. 

Streptomyces griseus 
II. Sporophores straight, verticils produced. 
1. Cottony aerial mycelium white, light tan, 
or pale pink. 
a. Soluble pigment on 
brown. 


bo 


protein media 
Streptomyces reticult 
b. No soluble brown pigment on protein 
media. 
Streptomyces hachijoensis 
III. Sporophores spiral-shaped. 
1. Predominantly closed spirals produced. 
a. Aerial mycelium gray. 
al. Soluble brown pigment on protein 
media. 
a2. Growth yellowish- 
brown, brown, or deep brown. 


colorless, 


Streptomyces olivochromogenes 
b?. Growth light purple to purplish- 
black. 
Streptomyces — purpureochro- 
mogenes 
b!. No soluble brown pigment. 
Streptomyces aureofaciens 
b. Aerial mycelium pale pink. 
al. Soluble pigment on protein media 
brown. 
Streptomyces lavendulae 
b!. No soluble brown pigment. 
Streptomyces fradiae 


i) 


. Predominantly open spirals or compact 
spirals produced. 
a. Aerial mycelium white. 
al. Soluble pigment on protein media 
brown. 
a®. Growth reddish. 
Streptomyces ruber 
bt. No soluble brown pigment. 
Streptomyces farinosus 
b. Aerial mycelium gray. 
al. Soluble pigment on protein media 
brown. 
a®, Abundant compact spirals pro- 
duced on aerial hyphae. 
Streptomyces naganishir 
b?. Growth colorless, white, light 
yellow, or yellowish-brown. 
Streptomyces diastatochromo- 
genes 
c?. Growth colorless, reddish-or- 
ange, or reddish-purple. 
Streptomyces griseoruber 
bt. No soluble brown pigment. 
a®. Growth colorless to creamy. 
Streptomyces albus 
b?. Growth colorless, white, light 
yellow, or yvellowish-brown. 
G 167 
myces Cacaor) 
ce. Growth colorless, light yellow, 
or ight pinkish-brown. 


(resembling Streplo- 


Streptomyces albogriseolus 
d?. Growth purple to pink to red. 
Streptomyces californicus 
ce. Aerial mycelium gray, but on certain 
media with dark, glistening 
patches. 


moist 


Streptomyces hygroscopicus 
IV. No characteristic features of aerial hyphae. 
1. Very limited aerial mycelium production 
on various media. 
a. Soluble pigment on protein media deep 
brown. 
al. Growth yellow to yellowish-brown. 
Streptomyces flavochromogenes 
b. Soluble pigment faint yellowish-brown. 
Streptomyces thioluteus 
2. Aerial mycelium white. 
a. Growth colorless, light yellow, or red- 
dish-orange. 
Streptomyces ruber 


4, BALDACCI SYSTEM (1956, 1958, 1959) 
Following the example of Sanfelice and 


Waksman, Baldacci divided the genus 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 


Streptomyces into sections, based upon the 
color of the substrate mycelium. Kach sec- 
tion was divided into series, on the basis of 
the color of the aerial mycelium. Each series 
was divided into species. 

The genus was characterized by the pres- 
ence or absence of spores, the arrangement 
of spores, and the ramification and breaking 
up of the substrate mycelium. The species 
were characterized by enzymatic reactions, 
antibiotic activity, and soluble pigments 
spreading through the substratum, depend- 
ing on nutrition and pH. 

Although Baldacci discussed the genus 
under the name ‘‘Actienomyces,” he actually 
meant Streptomyces, without recognizing it 


as such, since he made no mention of other 


species and other genera. 

A. Actinomycetes cum sporophora_ solitaria 
vel congregata. 

Section I. Substrate mycelium 
scant development on agar, showing a 

cobweb-like appearance; limited 


colorless; 


veiled 
sporulation. 
1. Aerial mycelium white. 
Series Albus 
2. Aerial mycelium sea-green. 
Series Griseus 
3. Aerial mycelium green-azure. 
Series Viridis 
4. Aerial mycelium azure. 
Series Caeruleus 
5. Aerial mycelium white-wine-lavender. 
Series Lavendulae 
6. Aerial mycelium light pink. 
Series Ioseus 


~J 


Aerial mycelium gray. 
Series Diastaticus 

II. Substrate 

development generally abundant on agar 


Section mycelium colored; 
(creamy, lichenoid, ete.); delayed or par- 
tial sporulation. 

(a) Substrate mycelium yellow to yellow- 

brown. 
9. Aerial mycelium white. 
Series Albidoflavus 


(b) 


(d) 


(e) 


95 


10. Aerial 
pink). 


mycelium pink (white to 
Series Roseoflavus 
11. Aerial mycelium yellow 
spots. 


with gray 


Series Flavus 
12. Aerial mycelium grayish. 
Series Aureus 
Substrate mycelium yellow to 
vellow. 


ereen- 


13. Aerial mycelium gray-white. 
Series Flavoviridis 
14. Aerial mycelium white to lemon- 
vellow. 
Series Virgatus 
Substrate mycelium yellow with green 
and pinkish spots. 
15. Aerial mycelium white to pink. 
Series WJadurae 
Substrate mycelium brown to black. 
16. Aerial mycelium white to gray. 
Series Scabies 
17. Aerial mycelium red. 
Series Roseochromogenes 
18. Aerial mycelium yellow. 
Series Sulphureus 
19. Aerial mycelium gray. 

Series A ntibioticus 
Aerial mycelium grayish-flesh-col- 
ored. 

Series Griseoincarnatus 
Vegetative mycelium brown to green- 
brown. 

21. Aerial mycelium gray. 
Series [ntermedius 
Vegetative mycelium brown. 
22. Aerial mycelium white to leather- 
brown. 
Series Rimosus 
Vegetative mycelium orange. 
23. Aerial mycelium seashell pink. 
Series Fradiac 
Vegetative mycelium flesh-rose. 
24, Aerial mycelium white. 
Series Bostroemi 
Vegetative mycelium red. 


96 THE ACTINOMYCETES, Vol. II 


25. Aerial mycelium white to pink. 
Series Albosporeus 
26. Aerial mycelium ash-gray. 
Series Cinereo-ruber 
(j) Vegetative mycelium violet-blue-red. 
. White to gray aerial mycelium with 
different shades. 


_ 


27 


Series Violaceus 
B. Actinomycetes cum sporophora opposita 
et verticillata* 
Section I. Substrate mycelium colorless. 
1. Aerial mycelium white or whitish. 
Series Circulatus 
2. Aerial mycelium gray and pinkish. 
Series Griseocarneus 
Section IT. Vegetative mycelium colored. 
(a) Substrate mycelum  lemon-yellow- 
creamy-colored. 
3. Aerial mycelium cinnamon-colored. 
Series Cinnamoneus 
(b) Substrate mycelium brownish-yellow. 
4. Aerial mycelium gray. 
Series Reticulr 
(c) Substrate mycelium brown. 
5. Aerial mycelium greenish-gray. 
Series Verticillatus 
(d) Substrate mycelium red. 
6. Aerial mycelium pinkish-red. 
Series Rubrireticuli 


5. GAUSE ET AL. SYSTEM (1957) 


Gause et al. modified Baldacci’s system, 
leaving out the sections and combining the 
pigmentation of the aerial mycelium with 
that of the substrate growth for series char- 
acterization. Each series was subdivided, on 
the basis of formation of a soluble pigment 
in a complex organic medium, or of the 
structure of the sporophores, or of the pig- 
mentation of a synthetic medium. These 
investigators, like Baldacci, adhered to the 
genus designation Actinomyces, without, 
however, considering the accumulated in- 
concerning all other 


formation genera. 


* These sections were placed by Baldacci in a 
separate genus ‘‘Streptoverticillium.” 


Little consideration was given to previously 
hamed species. 
I. Aerial mycelium rose-purple, 
strate mycelium colorless. 
Series Lavendulae-roseus 
II. Aerial mycelium rose-colored; sub- 
strate mycelium yellow. 
Series Fradiae 


sub- 


III. Aerial mycelium rose-colored; sub- 
strate mycelium brown. 
Series Fuscus 
IV. Aerial mycelium light rose; substrate 
mycelium violet: 
Series Roseoviolaceus 
V. Aerial mycelium rose-colored; sub- 
strate mycelium red. 
Series Ruber 
VI. Aerial mycelium yellowish-green or 
cream-colored; substrate mycelium 
colored or colorless. 
Series Helvolus 


VII. Aerial mycelium white; substrate 
mycelium colorless. 
Series Albus 
VIII. Aerial mycelium white; substrate 


mycelium red or brown. 
Series Albosporeus 
IX. Aerial mycelium blue or greenish- 
blue; substrate mycelium colorless or 
blue-colored. 
Series Coerulescens 
X. Aerial myceluum = gray; 
mycelium colorless. 
Series Griseus 
XI. Aerial mycelium gray, then black 
(result of autolysis); substrate my- 


substrate 


celium colorless. 
Series Nigrescens 

XI. Aerial mycelium gray; substrate my- 
celium yellow or orange. 

Series Aureus 
Aerial mycelium gray; substrate my- 
celium yellow-brown. 

Series Chrysomallus 
Aerial mycelium = gray; 
mycelium brown-black. 


XIII. 


substrate 


EV 


Series Chromogenes 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 97 


Aerial mycelium gray; substrate my- 
celium blue-violet or red-brown. 
Series Violaceus 


6. PRIDHAM, HESSELTINE, AND 


SYSTEM (1957, 1958) 


In their earlier system, these investigators 
divided the genus Streptomyces into seven 
groups. Each group was characterized by a 
distinct morphology of the sporophores in 
mature cultures, and by a distinct color of 
the aerial mycelium. This system  subse- 
quently was revised. Morphological sections 
and color series were established and, on the 
basis of literature study and laboratory in- 
vestigations, many species and known anti- 
biotic-producing strains were cataloged. It 
was suggested that evaluation of the compo- 
nent strains in the sections and series, by 
physiological tests, would allow the deter- 
mination of ranges of variation and a more 
logical approach to speciation in the genus. 
The placement by these investigators of 
strains in morphological sections, regardless 
of species designation, has suggested synon- 
ymy, as well as misidentification of many 
strains. 

The following bases were considered in 
justifying these subdivisions: 

1. The morphology of the sporophores of 
a particular strain does not appreciably 
change on substrata that support optimal 
formation of aerial mycelium, sporophores, 
and spores. Morphological patterns exhibited 
by streptomycetes are not subject to con- 
siderable variation, unless degeneration of a 
particular strain has occurred through im- 
proper maintenance. Morphological exami- 
nations should be made after two weeks’ 
incubation at 28-30°C on several appropriate 
media. 

2. The color of the sporulating aerial my- 
celium of a given strain at maturity was said 
not to differ appreciably from medium to 
medium. Each morphological section of the 
genus can be further subdivided into color 
“series.’? Each color series can be subdivided, 


BENEDICT 


on the basis of physiological criteria, into 


‘species.”’ Additional delineation can then 
be used to create ‘“‘varieties”’ or “physiolog- 
if need be. 


present 


ical forms,” 

3. The the 
Streptomyces is interpreted rather broadly. 
Some of the strains identified as members of 


concept of 


genus 


the genus may in reality belong to other 
genera. 

The proposed sections were designated as 

follows: 

I. Rectus-flexibilis 
straight, 
spirals. Type species S. 

Il. Retinaculum-apertum 


(RF). 
flexuous, or 


Sporophores 
fascicled; no 
Griseus. 
(RA). 
phores in the form of hooks, open loops, 
or greatly extended — spirals. 
species S. fradiae. 


Sporo- 
Type 


III. Spzra (S). Sporophores either short and 
gnarled, or in the form of compact 
spirals or of extended long and open 
spirals. Type species SS. viridochro- 
mogenes (lig. 26). 

IV. Monoverticillus (MV). Sporophores in 
the form of primary verticils attached 
to long, straight branches; no spirals. 

V. Monoverticillus-spira (MYV-S). 
phores as primary verticils attached to 


Sporo- 


long, straight branches; elements of 
verticils spiraled. 
VI. Biverticillus (BV). Sporophores as com- 


pound verticils attached to long, 
straight branches; no spirals. Type 


species S. cinnamomeus ft. cinnamomeus. 
Biverticillus-spira — (BIV-S). 
phores as compound verticils attached 
to long, straight branches; elements of 
secondary verticils spiraled. 

In addition to the above sections, another 
section was set up to include strains for 


Vat: 


Sporo- 


which no micromorphological data were 
available. 
Each ‘section’? was subdivided into 


“series”? based on the color of sporulating 
aerial mycelium at maturity. The proposed 
series were designated as follows: 


1. White. 


98 THE ACTINOMYCETES, 


Wolk ant 


Fraure 26. Spiral formation by Streptomyces 240 (Reproduced from: Naganishi, H. and Nomi, R. 


J. Fermentation Technol. 32: 492, 1954). 


2. Olive-buff (buff to tan to olive-buff). 
3. Yellow. 
. Blue (blue to blue-green to green). 

5. Red (pink to red to lavender to laven- 
der-gray). 

6. Gray (light gray to mouse-gray to 
brown-gray to gray-brown). 

An additional ‘unknown’ series was set 
up to include strains for which no color data 


i 


were available. 


7. ETTLINGER, CORBAZ, AND HUTTER SYSTEM 
(1958) 


Ettlinger ef al. considered four major 
characters of Streptomyces that were stable 
and reliable enough to justify their svstem of 
classification. These characters were: (a) 
morphology of the spores, (b) color of aerial 
mycelium, (¢) morphology of aerial myce- 
lium, and (d) formation of melanoid pig- 
ment. 

These investigators suggested combination 
of sections 4 and 6, and sections 5 and 7 of 
the Pridham et al. (1958) system, since they 
had never observed nonbranching verticils. 
They recognized a total of 15 morphological 
types distributed among the five sections. 


They also recognized the following color 
groups for the aerial mycelium: (1) niveus 
(snow-white), (2) (yellowish- to 
greenish-gray), (3) azureus (sky-blue), (4) 
cinnamoneus (light carmine to brownish), 
(5) cinereus (ash-gray), (6) prasinus (leek- 
green). 

They observed certain constant relation- 
ships among some of the four basic prop- 
erties. The griseus and cinnamoneus color 


Griseus 


groups were found to occur only in strains 
with smooth spores. The azureus and pra- 
sinus color groups occurred only in strains 
with spiny or hairy spores. The latter always 
were found associated with the occurrence 
of spirals. 

Other properties, such as soluble pigment 
on synthetic media, antibiotie activity, and 
pigmentation of substrate mycelium, were 
found to be variable. Gelatin liquefaction, 
milk coagulation, starch hydrolysis, and 
other physiological properties were not con- 
sidered of great value from a systematic 
point of view, since no true negative gelatin 
liquefaction or negative starch hydrolysis 
was ever detected. 

On the basis of the above properties, the 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 99 


following system of classification Was pro- 
posed for the genus Streptomyces: 


A. Spores spiny or hairy. 
I. Aerial mycelium blue. 
1. Streptomyces virido- 
chromogenes 
II. Aerial mycelium not blue. 
1. Aerial mycelium white. 


2. Streptomyces  pur- 
purascens 
2. Aerial mycelium not white. 
a. Aerial mycelium green. 
al. Spores with short spines. 
3. Streptomyces  pra- 


sinus 
b!. Spores with longer spines or with 
hair. 
a®. Spores with stiff spines. 
4. Streptomyces hirsu- 
tus 
b?. Spores with flexible hair. 
5. Streptomyces 
sinoptilosus 
b. Aerial mycelium gray. 
al. Sporophores in verticils. 
a®. Sporophores in open spirals; 
no melanin formation. 
6. Streptomyces nour- 
sev* 


pra- 


b?. Sporophores in closed spirals; 
melanin produced. 
7. Streptomyces echi- 
natus* 
b!. Sporophores not in verticils. 
a®. Sporophores in closed spirals. 
8. Streptomyces  albo- 
griseolus 
b?. Sporophores in open spirals. 
a®. Spirals irregular. 
9. Streptomyces  mac- 
ros poreus 
bs. Spirals regular. 
a', Spores spiny. 
10. Streptomyces grise- 
oflavus 
b*. Spores hairy. 
a°. Spirals with >5 turns; 
melanin-positive. 
11. Streptomyces  pilo- 


SUS 


* The verticillate nature of these organisms is 
open to question. 


FIGuRE 27. Sporogenous coiled hyphae of Strep- 
tomyces T 3110; taken from a gray area of a colony 
(Reproduced from: Duggar, B. M. ef al. Ann. 
N. Y. Acad. Sci. 60: 71-85, 1954). 


b®. Spirals with <5 turns, 
melanin-negative. 
12. Streptomyces flaveo- 
lus 
B. Spores smooth. 
I. Aerial mycelium yellowish- to greenish- 
gray. 
1. Melanin-negative. 
13. Streptomyces — gri- 
seus 
2. Melanin-positive. 
14. Streptomyces michi- 
ganensis 
Il. Aerial mycelium white. 
1. Sporophores in verticils. 
15. Streptomyces rubri- 
reticult 
2. No verticils produced. 
a. Sporophores form spirals. 
16. Streptomyces nive- 
oruber 
b. Sporophores straight or wavy. 
al. Melanin-negative. 
17. Streptomyces fulvis- 
STMUS 
b!. Melanin-positive. 
18. Streptomyces 
phaeochromogenes 


LOO THE ACTINOMYCETES, Vol. II 


IIT. Aerial mycelium hght carmine to brown- 
ish. 
1. Sporophores in verticils. 
a. Sporophores straight or wavy. 
19. Streptomyces ne- 
tropsis 
b. Sporophores in spirals. 
20. Streptomyces tendae 
2. Sporophores not in verticils. 
a. Sporophores straight. 
21. Streptomyces vene- 
zuelae 
b. Sporophores in spirals. 
al, Spirals at end of long, straight 
sporophores; melanin-positive. 
22. Streptomyces laven- 
dulae 
b!. Spirals different; melanin-nega- 
tive. 


a®. Spirals closed. 
23. Streptomyces — vio- 
laceoniger 
b?. Spirals open. 
a’. Spirals regular, usually 
>5 turns. 
24. Streptomyces — fra- 
diae 
b*. Spirals irregular, usually 
<5 turns. 
25. Streptomyces ery- 
thraeus 
IV. Aerial mycelium ash-gray. 
1. Sporophores in verticils. 
26. Streptomyces — reti- 
cult 
2. Sporophores not in verticils. 
a. Sporophores straight or wavy. 


& 


FIGURE 28. Sporogenous coiled hyphae of Streptomyces T 3110; taken from a blue sector of a colony 
(Reproduced from: Duggar, B. M. et al. Ann. N. Y. Acad. Sci. 60: 71-85, 1954). 


GROUPS AND SPECIES OF 


al, Sporophores sympodially 
branched. 
27. Streptomyces — viri- 
dogenes 
. Sporophores monopodially 
branched. 
2. Sporophores as side branches 
of sterile hyphae. 


28. Streptomyces ramu- 


es) 


« 
c 


losus 
2. Sporophores different. 
a’, Melanin-negative. 
29. Streptomyces oliva- 
ceus 
b’. Melanin-positive. 
30. Streptomyces 
bioticus 
b. Sporophores in spirals. 
al. Spirals closed. 
31. Streptomyces hygro- 
SCOptcus 


antt- 


b!. Spirals open. 
a®. Spirals irregular, usually >5 
turns. 
32. Streptomyces aureo- 
faciens 
b?2. Spirals regular, <5 turns. 
a’. Melanin-negative. 


33. Streptomyces — par- 
vullus 
bs. Melanin-positive. 
34. Streptomyces  gali- 


laeus 
8. SHINOBU SYSTEM (1958b) 


Shinobu proposed the following system 
pro} £ Sy} 

for grouping of the species of the genus 
Streptomyces: 


Group I. Monopodial branching, straight or wavy 
aerial mycelium; never producing spirals. 
Subgroup 1. Tyrosinase reaction: positive; ni- 

trite production: positive. 
Streptomyces olivaceus 


~ 


positive ; 
nitrite production: negative. 
Streptomyces phaeopurpureus 


Subgroup 2. Tyrosinase reaction: 


Ow 


reaction: negative; 
nitrite production: positive. 
Streptomyces sp. No. 2 
Tyrosinase reaction: negative; 
nitrite production: negative. 
Streptomyces sp. No. 232 
Group IL. Spiral formation; long or short, loose or 
compact, and open or closed spirals. 


Subgroup 3. Tyrosinase 


Subgroup 4. 


‘ GENUS STREPTOMYCES 10] 


Subgroup 5. Tyrosinase reaction: positive; 


nitrite production: positive. 
Streptomyces viridochromo- 
genes 
Subgroup 6. Tyrosinase’ reaction: positive; 
nitrite production: negative. 
Streptomyces sp. No. 2076. 
reaction: 


. Tyrosinase negative; 


“I 


Subgroup 
nitrite production: positive. 
Streptomyces sp. No. 236 
negative; 
nitrite production: negative. 
Streptomyces scabies 


Subgroup 8. Tyrosinase reaction: 


Verticil formation; primary and 
verticils; rarely one tertiary verticil 


Group III. 
secondary 
(Fig. 29). 
Subgroup 9. Tyrosinase reaction: positive; 

nitrite production: positive. 

Streptomyces hiroshimensis 
Tyrosinase reaction: positive; 
nitrite production: negative. 

Streptomyces luteoverticillatus 

Tyrosinase reaction: negative; 

nitrite production: positive. 


Subgroup 10. 


Subgroup 11. 
Streptomyces  roseoverticilla- 
tus 
reaction: negative; 
nitrite production: negative. 
Streptomyces olivoverticillatus 
Group IV. Intermediate group of Nitella-type and 
Anitella-type verticil. 
Subgroup 13. 


Subgroup 12. Tyrosinase 


Streptomyces spiroverticillatus 


Shinobu further emphasized that in the 
identification of species of Streptomyces other 
characteristics should be considered. These 
are the following: 

1. Morphological properties: type of colony, 
shape of spiral, etc. 

2. Physiological 
nase, and amylase reactions; utilization of carbon 


properties: cellulase, man- 
and nitrogen sources, etc. 

3. Cultural properties: growth of colony, pro- 
duction of pigment, ete. 


9. FROMMER SYSTEM (1959) 


This system does not apply to the genus 


Streptomyces as a whole but only to the 


actinomycin-producing species. 
A. Chromogenic group. 


I. Spirals not formed on aerial mycelium. 
Occasionally a few spirals are found. 


102 


THE ACTINOMYCETES, Vol. II 


Ficure 29. Verticil formation (Nitella type) including both primary and secondary verticils (Re 
produced from: Shinobu, R. Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sei. 7, 1958). 


1. Aerial mycelium on_ synthetic 
media gray. Tyrosinase-negative. 
Streptomyces antibioticus 


mycelium on 


agar 


bo 


Aerial synthetic 
media yellow. Tvrosinase-positive. 


agar 


Streptomyces michiganensis 
Il. Numerous spirals produced on aerial my- 
celium. 
1. Yellow or yellow-green pigment 
duced on synthetic agar. 


pro- 


Streptomyces galbus 
2. Soluble pigment on synthetic agar, dark 
brown. 
Streptomyces lanatus 
B. Nonchromogenic group. 
I. No spirals on aerial mycelium. 
1. Strongly proteolytic. Aerial mycelium 
on synthetic agar white, yellow, or 
greenish. 
Streptomyces chrysomallus 
2. Weakly proteolytic. Aerial mycelium on 
synthetic agar mouse-gray. 
Streptomyces chrysomallus v. 
fumigatus 
II. Numerous spirals on aerial mycelium. 
1. Aerial elycerol-glycine 
agar grayish-rose. Practically no growth 


mycelium on 


on synthetic agar. 
Streptomyces murinus 


faciens, S. 


2. Abundant growth on synthetic agar. 
Aerial mycelium on 
agar cream-colored. 


glycerol-glycine 


Streptomyces galbus v. achro- 
mogenes 


10. MAYAMA SYSTEM (1959) 

Mayama (1959) concluded that morphol- 
ogy, and types. of 
growth on liquid media are the most impor- 


serological reactions, 


tant properties for the classification of 
Streptomyces. On the basis of these prop- 
erties, he divided the genus into seven 


groups: S. olivaceus, S. lavendulae, S. aureo- 
griseolus, S. albus, S. rimosus, and 
S. reticult. In addition to these, he also listed 
a number of species for which no group char- 
acteristics were known, notably S. anti- 
bioticus, S. fulvissimus, S. ruber, S. coelicolor, 
etc. 

Mayama (1959) and Mayama and Ta- 
wara (1959) classified the genus Strepto- 
myces into five sections and 14 series: 
Section I. Aerial mycelium — irregularly 

branched. Sporophores produced at the 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 


FrGureE 30. Spore formation in Streptomyces species (Reproduced from: Shinobu, R. Mem. Osaka 


Univ. Lib. Arts and Ed. B. Nat. Sei. 7, 1958). 


terminal portion of the branching hyphae. 
Series 1. Sporophores straight to flexu- 
ous. 
Series 2. Sporophores form open loops. 
Series 3. Sporophores form spirals. 
Section II. Aerial mycelium branches in tuft 
formations. Sporophores produced at the 
terminal portion of the branching hyphae. 
Series 4. Sporophores straight to flexu- 
ous. 
Series 5. Sporophores form open loops. 
Series 6. 
Section ITI. 


main stem. Sporophores produced at the 


Sporophores form spirals. 
Aerial mycelium forms long 


terminal portion of side branches. Non- 

verticillate. 

Series 7. Sporophores straight to flexu- 
ous. 

Series 8. Sporophores form open loops. 

Series 9. Sporophores form spirals. 


Section IV. Aerial mycelium forms long 
main stem. Sporophores produced at 


terminal portion of side branches. Ver- 
ticillate. 


Series 10. Monoverticillate, straight to 
flexuous. 
Series 11. 


Series 12. 


Monoverticillate, spirals. 
Biverticillate, straight to flexu- 
OuS. 
Series 15. 
Section V. 
Series 14. 


Biverticillate, spirals. 


No aerial mycelium. 


11. NOMI SYSTEM 

Finally a purely morphological system 
may Nomi (1959) proposed a 
division of the genus Streptomyces into eight 


be listed. 


morphological groups. He returned to an 
earher concept of Drechsler (1919) that the 
nature of the turn of the spirals, namely 
sinistrorse and dextrorse, is an important 
characteristic of Streptomyces species. He 
recognized, however, that some cultures may 
be rather indefinite in this respect. 


A. Aerial hyphae somewhat flexuous or straight; 
few long hyphae. The terminal filaments de- 
velop into spiral-shaped sporophores. 

1. Spirals sinistrorse. 


104 THE ACTINOMYCETES, Vol. II 


re 


te ME SR 
Y ob 


10 11 12 13 


oO 
Puiate III. Morphological groups in the genus Streptomyces (Mayama, 1959) (For details, see text, 
pp. 102-103). 
Section I: series 1—3 
Section IT: series 4—6 
Section IIT: series 7—9 
Section IV: series 10—18 


B: 


D. 


ee 


GROUPS AND SPECIES OF 


a. Spirals long, extended to compact. 


S. coelicolor, S. albogriseolus, S. fla- 
veolus, S. parvullus 
b. Spirals compact to compressed. None 


found. 
2. Spirals dextrorse. 
a. Spirals long, extended to compact. 
S. viridochromogenes 
b. Spirals compact to compressed. 
Streptomyces sp. No. 189 
Most aerial hyphae long, straight or slightly 
flexuous. They do not sporulate, but give rise 
to short side branches whose terminal filaments 
develop into spiral-shaped sporophores. 
1. Spirals sinistrorse. 
S. purpurascens 
2. Spirals dextrorse. 
Various unidentified forms. 


. Aerial hyphae irregularly flexuous or wavy; 


long hyphae absent. Terminal filaments form 
spirals. 
1. Spirals sinistrorse. 
S. sulphureus 
2. Curvature of spiral indefinite. 
S. griseoluteus 
Aerial hyphae long, straight, or wavy. They 
give rise to short side branches, which develop 
into spore-bearing hyphae containing spirals. 
1. Spirals sinistrorse. 
S. hygroscopicus, S. violaceoniger, S. 
albus 


. Aerial hyphae in clusters. The terminal fila- 


ments develop into sporophores, both spiral 
and nonspiral forming. 
S. vinaceous, S. microflavus, S. fra- 
diae, S. lavendulae, S. 
roseochromogenes, 


virginiae, S. 
cinnamonensis, S. 
S. phaeochromogenes 
Aerial hyphae branch in clusters. No spirals or 
loops. 
S. venezuelae, S. tanashiensis, S. 
bikiniensis, S. antibioticus, S. aureo- 
faciens, S. olivaceus, S. nitrosporeus, 
S. griseus, S. lipmanii, S. rutgersensis, 
S. parvus, S. flavovirens, S. californi- 
cus, S. vinaceus, S. ruber, S. caeruleus 
Aerial hyphae long, straight or slightly flexu- 
ous. Verticillate. No spirals. 
S. reticuli, S. griseocarneus, S. echi- 
mensis, S. hiroshimensis, S. salmoni- 
cida, S. thioluteus, S. albireticuli, S. 
netropsis 


. Aerial hyphae somewhat flexuous or wavy. 


Long hyphae and spirals are not produced. 
S. albus (atypical), S. halstedii, S. 


GENUS STREPTOMYCES 105 


scabies, S. verne, S. griseolus, S. 


erythreus (Plates IV and V) 


12. OTHER SYSTEMS 

Other systems have been proposed for the 
the 
Some of these systems are modifications or 
supplementations of that presented in Ber- 
gey’s Manual (7th ed., 1957), or modifica- 
tions of one or the other of those outlined in 


classification of genus Streptomyces. 


this chapter. 

One of these is the system outlined by 
Routien (1959). The various species in- 
cluded in the Streptomyces 
divided into three major groups: (1) sapro- 
phytes; (2) plant parasites or cultures 
isolated from diseased plants or from soil in 
which diseased plants were grown; (3) cul- 
tures isolated from animal tissues. These 


genus were 


groups were subdivided on the basis of for- 
mation and color of aerial mycelium (green, 
brownish-purple to black, blue-gray or blue- 
green, yellowish to orange, pink to rose, ete.). 
The color of the substrate mycelium and the 
various biochemical properties were then 
used for further subdivisions. Morphology 
(spiral formation, shape of spores) played 
only a minor role in this system. See also 
Sakai, 1959. 


Summary of the Properties Used in 
Subdividing the Genus 
Streptomyces 


Evaluation of the above systems of clas- 
sification leads to the conclusion that sporo- 
phore morphology has been given first or 
second consideration by the great majority 
of investigators. Lesser attention was paid 
to the color of the aerial mycelium and the 
nature of soluble pigments. Chromogenesis, 
or pigment formation in protein media, Was 
often given first position. Antibiotic pro- 
duction and ecology received the least con- 
sideration. 

In Baldacci’s system of dividing the genus 
into groups or series, the color of the sub- 


THE ACTINOMYCETES, Vol. II 


106 

e ‘ 

a . 

y A 

ie a) ‘J 

| 

er ae 
2x) 

Q 2 


Puate IV. Morphological types, according to Nomi (1959). a. represents a schematic presentation 


of each type; b. gives the actual photograph. 
A. Aerial hyphae flexuous or straight; spirals extended to compact (S. coelicolor). 
B. Aerial hyphae straight; spirals on side branches (S. purpurascens). 

(See continuation, next plate). 


GROUPS AND SPECIES OF GENUS STREPTOMYCES LO7 


PLate IV. (Continued) 
C. Aerial hyphae wavy; spirals on terminal filaments (S. griseoluteus). 
D. Aerial hyphae long, straight or wavy; sporophores as side branches; spirals produced (S. hygroscopi- 


cus). 


108 THE ACTINOMYCETES, Vol. II 


ae 
ine 


a 
oi 
{ 

é 


om 


Ly. 


- 

fi 
; 
4 
y 


PLtate V. Morphological types, according to Nomi (1959). a. represents a schematic presentation of 
each type; b. gives the actual photograph. 
i. Aerial hyphae in clusters, terminal filaments developing into sporophores, both spiral- and nonspiral- 
forming GS. lave ndulae, Se roseochromogenes ). 
F. Aerial hyphae in clusters; no spirals or loops (S. antibioticus). 
(See continuation, next plate). 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 


ef, 
lvl 


30 


Vv 


50 20 
| Nvvreluvistta quali 


Prats V. (Continued) 
G. Aerial hyphae verticillate; no spirals (S. hiroshimensis). 


H. Aerial hyphae flexuous or wavy; no long hyphae and no spirals (S. albus). 


109 


L10 


strate mycelium was taken as the basis for 
the primary subdivision into sections, and 
the color of the aerial mycelium for the 
secondary subdivision into series. In the 
systems used by Flaig and Kutzner (1954) 
and by Ikutzner (1956), the color of the 
aerial mycelium was used in connection with 
that of the substrate mycelium. The system 
proposed by Yamaguchi and Saburi (1955) 
was based principally upon the structure of 
the sporophores, the color of the aerial my- 
celium being utilized in a secondary sub- 
division; in the final characterization, ad- 
vantage was taken of the production of 
soluble pigments. A similar system was used 
by Shinobu (1958). Krassilnikov (1941, 
1949), Hesseltine ef al. (1954), Pridham e¢ al. 
(1958), Etthnger et al. (1958), Mayama 
(1959), and Nomi (1959) used morphological 
criteria for the primary subdivision of the 
genus. 

Each one of the above systems has in it- 
self certain serious limitations. It 1s neces- 
sary, therefore, to combine several prop- 
erties in order to bring out the characteristics 
of the group or series, and especially those of 
the species. 


Proposed System of Classification of the Genus 
Streptomyces into Groups or Series 


In presenting the following system, full 
cognizance is taken of the criticisms to be 
directed against it, especially that the for- 
mation of the melanin pigment is given 
leading consideration, and that the pro- 
duction of other soluble pigments as well as 
of antibiotics is also given important con- 
sideration. I have felt that because of my 
own previous proposals, especially those in- 
corporated in the various editions of Bergey’s 
Manual, and my own interest in antibiotics, 
the best I could do would be to modify this 
system slightly. I hope that it will serve its 
purpose in the future as it has done in the 
past. The suggested series further broaden 


my earlier concept of species-groups. In 


THE ACTINOMYCETES, Vol. II 


view of the fact, however, that it is desirable 
for each group to be designated by a repre- 
sentative species, it has been found neces- 
sary, In some cases, to use a more recent, 
well defined species rather than one used long 
ago, for which no well established species is 
now recognized. This is true, for example, of 
the “‘chromogenes” series, which has been 
designated as Phaeochromogenes, for which a 
well recognized type culture is available. 

I beheve that the system of classification 
of the genus Streptomyces into series proposed 
here is simple and convenient. The use of 
ecological properties as a basis for the major 
subdivision of the genus, as in the last edi- 
tion of Bergey’s Manual, has been discarded. 
The thermophilic forms have been, for the 
part, transferred to other genera 
(Chapter 11). The animal and plant isolates, 
including both pathogens and saprophytes, 
have been distributed throughout the genus, 


most 


among the various series, where they logi- 
cally belong on the basis of their morphologi- 
eal, cultural, and biochemical properties. 
Both morphological (structure of sporo- 
phores) and cultural (color of aerial myce- 
lium, melanin formation) characters are 
combined in the major subdivision of the 
genus into subgenera and into series. Each 
series is subdivided, on the basis of specific 
cultural and biochemical properties, into 
species. Formation of soluble pigments, 
pigmentation, and antibiotic production are 
also frequently taken advantage of in char- 
acterizing species. To identify a new culture 
properly, it is important to consider not only 
the series subdivision and species classifi- 
cation, but also the detailed description of 
each organism. Before it can be decided 
whether a newly isolated culture is different 
from one already described, a study should 
also be made of the varieties within the 
species previously created, as well as possible 
mutations and variations within the culture. 
The names given for the various series are 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 


the names of the type species within the 
particular series. (See also Table 10.) 


Genus Streptomyces Waksman and Hen- 
rici, containing 16 series. Type species S. 
albus (Rossi-Doria) Waksman and Henrici. 

A. Subgenus Streptomyces Waksman, with 
14 series. Type species Streptomyces (Strepto- 
myces) albus (Rossi-Doria) Waksman and 
Henrici. 

B. Subgenus Streptoverticillium Baldacci, 
with 2 series. Type species Streptomyces 
(Streptoverticillium) reticuli (Waksman and 
Curtis) Waksman. 


A. Sporophores straight, wavy, or spiral- 
forming. Subgenus Streptomyces 
Subgroup A. MESOPHILIC 


I. Melanin-negative 


Series 1. Albus. This series is character- 
ized by a white to light gray aerial myce- 
lium, covering the whole of the substrate 
growth; concentric rings may be formed. It 
is melanin-negative. A faint brownish pig- 
ment may be produced on organic media. 
Sporophores are spiral-shaped, occasionally 
broom-shaped. The species within this series 
are usually strongly proteolytic, without 
formation of bad-smelling products. It may 
be argued that the type species S. albus is 
no longer available and that many species 
possess similar properties. This series and 
this species must be recognized historically, 
whatever the final the type 
species to be adopted (Pridham and Lyons, 
1960). 

Series 2. 
terized by a gray aerial mycelium, ranging 
in color from light gray to mouse-gray or 
smoke-gray to ash-gray to bluish-gray; it 
may be white at first, later turning various 
shades of gray. Substrate growth may be 


decision of 


Cinereus. This series is charac- 


colorless or yellowish, turning gray to dark. 
Frequently a soluble yellow pigment is pro- 
duced. The sporophores are either straight 
or spiral-shaped. 


Nel 


Series 3. Flavus. This series, as well, has a 
long historical background; it was one of 
the three groups so designated by Sanfelice 
in 1904. It is characterized by a yellow or 
vellow-orange to yellowish-brown substrate 
growth, and by an aerial mycelium which is 
white to yellowish to gray. A  yellowish- 
green to golden yellow soluble pigment. is 
usually produced. 
straight, or spiral-shaped. 

Series 4. Ruber. This series is character- 


Sporophores are long, 


ized by a pink to orange to red substrate 
growth, and by a white to yellowish to red 
aerial mycelium. No soluble pigment is pro- 
duced; occasionally a yellowish to brownish 
pigment may be formed. Sporophores are 
straight or spiral-shaped. 

Series 5. Viridis. This series is character- 
ized by a green to dark green substrate 
growth, and by a white to gray to light 
green aerial mycelium. Usually there is no 
soluble pigment; occasionally a light green 
pigment is formed. Sporophores are straight 
or spiral-shaped. 

Series 6. Violaceoruber. Substrate growth 
is at first colorless, gradually becoming red 
or blue; aerial mycelium is white to gray 
with bluish tinge. The characteristic soluble 
pigment is blue, frequently changing in color 
with the reaction of the medium; it is blue 
at an alkaline and red at an acid reaction. 
Sporophores form spirals. 

Series 7. Fradiae. This series is character- 
ized by a yellow to orange substrate growth, 
and by a powdery pink to seashell pink to 
light orange aerial mycelium. Usually no 
soluble pigment forms on synthetic or or- 
ganic media; a pink pigment may occasion- 
ally be produced. Sporophores are straight 
or spiral-shaped. Species are strongly pro- 
teolytic and antagonistic. 

Series 8. Griseus. This series is character- 
ized by colorless substrate growth, becom- 
ing, in certain media, brown to almost olive- 
black. Aerial mycelium is yellowish with a 
greenish tint, or greenish-gray or sea-green. 


112 


No soluble pigment is produced. Sporophores 
are straight or flexuous, producing tufts. 

Series 9. Hygroscopicus. This series is char- 
acterized by a colorless substrate growth, 
which gradually becomes yellow, dark to 
almost black. Aerial mycelium is white to 
gray; it is often moist and even soft. Sporo- 
phores are straight and spiral-shaped. No 
soluble pigment is produced. 


Il. Alelanin-positive 


Series 10. 
white to gray to buff. Substrate growth is 
brown to black. Sporophores are straight 
or spiral-shaped. 

Series 11. Lavendulae. Aerial mycelium is 


Scabies. Aerial mycelium is 


lavender to rose or pink to vinaceous laven- 
der. Substrate growth is colorless to cream- 
colored. Sporophores are not flexuous, often 
forming loops and loose or open spirals. 

Series 12. Hrythrochromogenes. Aerial my- 
celium is white with brownish shade. Sub- 
strate growth is brown to black. Sporo- 
phores produce spirals. 

Series 13. Viridochromogenes. Aerial my- 
celium is light green to olive-green. Sub- 
strate growth is grayish-green to brown to 
black. Sporophores produce spirals. 


Subgroup B. THERMOPHILIC 


Series 14. Thermophilus. This series com- 
prises six species. These are listed in Chapter 
1 


B. Sporophores produce verticils. 
Subgenus Streptoverticillium 
AMelanin-negative 


This and the 
next series are largely characterized by the 


Series 15. Cinnamomeus. 
morphological structure of their sporulating 
bodies. The sporophores produce verticils on 
the primary or on the secondary branches of 
the aerial mycelium, or on both. The spore 
chains are straight or spiral-shaped. This 


THE ACTINOMYCETES, Vol. II 


group is further characterized by being 
melanin-negative. The aerial mycelium is 
white to pinkish to cinnamon-colored. 


Melanin-positive 


Series 16. Reticuli. This series is charac- 
terized by the same morphological proper- 
ties as Series 15, but it is melanin-positive. 
The aerial mycelium is white to gray. 


There is a considerable overlapping of the 
different series. Frequently a given culture 
may be placed in one series or another, de- 
pending on the media and the conditions 
used for growing the organism, not to 
mention the idiosyncrasies of the observer. 
Classification becomes particularly difficult 
when one bears in mind the marked varia- 
tions frequently observed between different 
isolates of the same species, and the tendency 
of individual cultures to mutate upon con- 
tinued cultivation in artificial media. The 
fact that identification is frequently based 
upon comparison with published descrip- 
tions rather than with type cultures has 
resulted in the tendency to create new species 
on the basis of minor differences, some of 
which may be simple variations. 

Most of the series are made up of non- 
chromogenic forms (or those that produce no 
melanoid pigments), although some of the 
constituent species may produce faint brown 
soluble pigments on certain media. Some of 
these pigments result from lysis of the my- 
celium of the organism; others may be quite 
distinet and chemically different from the 
typical melanoid or chromogenic pigments, 
eg. the olive-green to olive-buff pigment 
frequently produced by S. griseus. Fewer 
series are composed of truly chromogenic 
forms, those capable of producing brown to 
dark brown or almost black soluble pig- 
ments with protein-containing media. 

The various ‘ 
quite distinct from those proposed by Bal- 
dacci et al. (1954). They proposed, for ex- 


‘series’? suggested here are 


GROUPS AND SPECIES OF GENUS STREPTOMYCES 113 


ample, a ‘‘Bostroem’’ series, for which no 
true representative can be recognized at 
present. Their series ‘‘Antibioticus’? and 
“Caeruleus”’ 
reasons that need not be discussed further 
here. Certainly, the idea expressed by Bal- 
dacei et al. in 1955 that “it is not possible 
to speak of a natural systemization of these 
microorganisms at the present 
knowledge... for the time being, one must 
limit oneself to a classification aiming solely 
at diagnosis and nomenclature,’ represents 
a defeatist attitude. It is well illustrated by 
his creation of a series named ‘‘Diastaticus.”’ 


state of 


Here were included pigmented and nonpig- 
mented organisms, chromogenic and non- 
chromogenic, with such fantastic names as 
A. rubrocyanodiastaticus, and such varieties 
as atrodiastaticus. This is certainly a good 
cause for confusion. 

Similar criticism 
grouping of the species proposed by Gause 
et al. (1957). Whereas Baldacci used the 
color of the substrate mycelium for primary 
subdivisions of the genus into sections, and 
the pigmentation of the aerial mycelium for 
the further division of the into 
series, Gause et al. (1957) omitted the sec- 
tions altogether, and divided the genus 
directly into series largely on the basis of 
the pigmentation of the aerial mycelium. 
Descriptions of 37 old and 71 new species 


‘an be apphed to the 


sections 


were reported by a group of six collabora- 
tors. The authorship was of a collective 
nature, with all the possibilities for confusing 
the credit to be assigned to each individual, 
since it is stated that “the study of the 
structure, classification, ecology and dis- 
the 
attention of large scientific collectives in a 


tribution of actinomycetes occupies 


number of institutes and universities.” 
Proceeding from the fact that so many new 
species have been recently created, in de- 
scribing producers of antibiotics, these in- 


vestigators assumed that this was further 


‘cannot be accepted for other 


proof that the old systems of classification 
were insufficient. Although it was recog- 
nized that the pigmentation of the aerial 
mycelium, the major criterion for classifica- 
tion purposes, could change on continued 
incubation, as in the case of their Group I, 
“cultures with lavender and brownish-rose 
pigment may change in color to salmon, red, 
and pale terra cotta,’ nevertheless, 15 series 
were adopted. This fact alone would tend to 
cast doubt upon the significance of recog- 
nizing major groups solely on the basis of 
pigmentation of the aerial mycelium. In 
establishing the species, structure of the 
sporophores was used in some cases; in 
others, the pigmentation of a single medium, 
frequently unknown in composition, was 
used.* To complicate the situation further, 
authors of old species and emendations of 
species were incorrectly credited, providing 
a potential source of confused nomenclature. 

Lieske (1921) was the classical ‘‘tumper,”’ 
largely because of the limitations imposed 
by the use of complex organic media, and 
because he was not aware of some of the 
characteristic morphological and cultural 
properties of the organisms, brought out 
particularly on synthetic media. The “‘split- 
ting” attitudes of Baldacci, Gause, 
certain others have brought the system of 
classifying this important group of organ- 


4 and 


isms to undesirable extremes. 

In the decision to classify the genus Strep- 
tomyces into 16 series, it is well understood 
that in time other series will be added; some 
of those presented here may eventually be 
spht into two or more series; some of the 
varieties may be raised to the status of 
species; or some of the species may be raised 
to the status of series. 

The problem of whether antibiotic produc- 
tion is a species characteristic is still un- 
settled. Undoubtedly, the 
different antibiotics can be combined with 


production of 


* Hottinger’s, to which no reference is given. 


114 THE ACTINOMYCETES, Vol. II 


certain other distinct properties, such as 
pigmentation, morphology, and carbon utili- 
zation, to justify the creation of new species. 
This has actually been done for the separa- 
tion of S. griseinus from S. griseus and the 


raising of the latter to a series status. In 
other cases, however, the mere formation of 
a different antibiotic without other accom- 
panying differences hardly justifies, for the 
present at least, the creation of new species. 


Chapter 6 


Series and Species of the Genus 


Streptomyces 


The genus Streptomyces was created in 
1943, to separate certain aerial mycelium- 
producing actinomycetes from the rest of 
the order Actinomycetales. Although there 
is considerable overlapping between species 
placed in this genus and those of Nocardia 
and some of the thermophilic groups, there 
are certain important properties that may 
said to characterize this genus, thus 
separating it, if not for any other reason 
than that of convenience, from the others. 

The major important characteristic prop- 
erties that distinguish the genus Streptomy- 
ces from the others can be briefly summar- 
ized as follows: 


be 


1. A more or less branched, nonseptate, 
substrate or vegetative mycelium (stroma) 
is produced. 

2. Growth takes place either on the sur- 
face of agar or gelatin media or penetrates 
deep into the medium, forming a compact, 
often leathery mass, designated as a colony. 
During growth in stationary liquid 
media, no turbidity is produced except on 


9 
oO. 


lysis; the masses of growth appear as clumps 
or compact Masses. 

4. The surface colony gradually becomes 
covered with an aerial mycelium, though 
this occasionally may not occur. 

5. The aerial mycelium produces sporo- 
genous hyphae or fruiting bodies, which are 
straight, or in the form of tufts, or curved, 
spiral-shaped, or verticillate. 


ibs) 


6. The sporophores carry chains of single- 
celled spores (or conidia), which vary in 
shape from spherical to oblong or cylindrical, 
and also in surface appearance when viewed 
with the electron microscope. 

7. The vegetative growth, the aerial my- 
celium, and the spores en masse frequently 
are colored in a characteristic manner; the 
color may also dissolve into the medium, 
producing a 


‘ 


‘soluble pigment.”’ 

8. The species are aerobic and meso- 
philic, nonacid-fast and gram-positive. 

The genus Streptomyces comprises, by far, 
the largest number of species of actinomy- 
cetes now known to occur in nature. The 
various species belonging to this genus differ 
in cultural, 
physiological, and biochemical properties. 


greatly their morphological, 
They include the majority of antibiotic- 
producing actinomycetes. The growing eco- 
nomic importance of these organisms has 
tended to increase the need for the separa- 
tion of the genus into groups, each of which 
would contain one or more species. This 
need has recently been further emphasized 
by the creation of numerous additional spe- 
cles. 

The color of the aerial and substrate my- 
celium, the morphology of sporophores, and 
the formation of melanin pigments have 
been largely used for the separation of the 
genus Streptomyces into series and species. 
lor the supplementary characterization of 


116 THE ACTINOMYCETES, Vol. II 


the species, the formation of nonmelanin 
pigments, the ecology of the organisms, and 
some of the biochemical properties (notably 
antibiotic formation) have been utilized. 
This is also true of their practical utiliza- 
tion for the production of enzymes, vita- 
mins, or antibiotics. 

It would appear that morphological prop- 
erties might offer a natural and stable basis 
for a system of classification of these organ- 
isms. Unfortunately, certain characteristic 
morphological features of the genus Strepto- 
myces undergo variation, depending upon 
the nutrition of the organisms and upon the 
environment. This tended to suggest, at 
first, the inadvisability of considering mor- 
phology as the major basis for the classifica- 
tion of the genus Streptomyces. This was true, 
for example, of Drechsler’s idea of consider- 
ing the type of curvature of the spiral-form- 
ing aerial hyphae as a basis for classification. 
It was also true of Waksman’s suggestion 
that the mode of branching of the sporo- 
phores might be used for this purpose. The 
ideas of Krassilnikov (1941, 1949) in empha- 
sizing the size and shape of the spore would 
also meet with similar criticism. Flaig et al. 
(1955), as well as Ettlinger et al. (1958), pro- 
posed use of the nature of the spore surface 
as a species characteristic; unfortunately, 
this property, depending as it does upon the 
use of the electron microscope, has not been 
readily enough established to enable the sep- 
aration of the genus into groups and species. 

Certain morphological properties are 
now, however, well recognized and can be 
utilized for the separation of certain groups 
of organisms belonging to the genus Strepto- 
myces. Such groups possess sufficiently well 
defined morphological features to differen- 
tiate them from the rest of the genus. This 
is true particularly of those forms that 
produce radiating sporulating hyphae (verti- 
cils), with straight or spiral-shaped branches 
on the main sporophores or on the side 
branches. This property makes it possible 


to distinguish these particular forms from 
the majority of other species of Streptomy- 
ces, Which produce either straight, flexuous, 
curved, or spiral-forming sporophores. Sev- 
eral systems of classification of the genus 
Streptomyces into series (Hesseltine ef al., 
1954; Shinobu, 1958b) took full advantage 
of the verticil-producing property; Baldacci 
(1959) went so far as to suggest placing the 
latter into a separate genus. The separation 
of the spiral-forming from the straight 
sporophore-producing types into separate 
groups has also been frequently suggested. 

In view of the above limitations, the only 
conclusion that can be reached is that, for 
the present at least, a logical system of 
separation of the genus Streptomyces into a 
number of distinct series should be based 
upon a combination of several of the mor- 
phological and physiological properties. It 
is proposed here to divide the genus into 
16 series. This system, likewise, is open to 
criticism: (a) there is left, for example, con- 
siderable room for a certain amount of over- 
lapping in some of the major properties which 
characterize the various series; (b) the posi- 
tion of a species within a series is not always 
well defined, and some of the species could 
frequently be placed with as much Justifica- 
tion in one series as in another; (c) there is, 
further, a lack of uniformity in characteriz- 
ing the various series: In some instances 
color of the aerial mycelium or of the sub- 
strate growth is used, and in others the 
formation of soluble pigments is emphasized. 

Fully recognizing the above limitations, 
however, I feel that a sound basis has been 
laid, taking full advantage of the knowledge 
now available, for dividing the genus Strepto- 
myces into series. As further information 
accumulates, the system can easily be modi- 
fied, since it lends itself readily to various 
changes and modifications. 

A detailed characterization of the various 
series 1s presented here. Some of the series 
are described in greater detail than others. 


SERIES 


This is due either to their longer historical 
background or to their capacity to form 
important economic products, especially an- 
tibiotics. 

In characterizing each series, the follow- 
ing properties have been given special con- 
sideration. 

a. Morphological properties, — notably 
structure of sporophores and spores. 

b. Color of aerial mycelium on synthetic 
media. 

ce. Formation of soluble brown pigment 
(melanin) on protein media. 

d. Spore surface. 

e. Other characteristic properties, such 
as color of substrate growth, formation of 


soluble, nonmelanoid 


pigments, rate of 
proteolysis, or production of specific anti- 
bioties. 

A summary of the properties of the 15 
series within the genus Streptomyces is given 


n Table 10. 


AND SPECIES OF 


GENUS STREPTOMYCES 117 


I. Series Albus 


Characteristic Properties 

a. Sporophores produce spirals; spores 
spherical to oval. 

b. Color of aerial mycelium white. 

c. Melanin-negative. 

d. No soluble pigment produced (except 
a faint brown pigment on certain media). 

e. Weakly proteolytic and weakly antago- 
nistic. 

The Albus series comprises a large num- 
ber of organisms, characterized by the pro- 
duction of 
substrate 


a typical leathery and compact 
growth, various 


media. Aerial mycelium is snow-white to 


colorless on 
white in color, assuming various shades as 
the culture grows older. The sporophores 
are long and form spirals; the spores are 
spherical to ovoid. The various strains grow 
well on both organic and synthetic media. 
They vary greatly in their proteolytic and 
diastatic properties. As a rule, this group of 


TABLE 10 


Characteristic properties of various series of Streptomyces 
SA Name of series Mee Aerial mycelium Color of growth forma Type species 
1 Albus — White Colorless + .§. albus 
2 Cinereus = White to gray Colorless to yel- | +— | S. craterifer 
low 
3 Flavus — Mouse-gray Yellow + | 8S. flavus 
4 Ruber — Rose Red +— | §. ruber 
5 Viridis —_ Gray to green Green +— | S. viridis 
6 Violaceoruber - Gray Red to blue + | S. violaceoruber 
7 Fradiae - Pink to rose Yellow to orange +— | S. fradiae 
8 Griseus — Grass-green Colorless to olive- — |S. griseus 
buff 
9 Hygroscopicus — White to gray Dark gray to black + |S. hygroscopicus 
10 | Scabies + Gray Brown to black + |S. scabies 
11 Lavendulae a Lavender Colorless +—  §. lavendulae 
12 Erythrochromo- + Yellowish Orange +— |S. erythrochromo- 
genes genes 
13 Viridochromo- + Green to olive- | Brownish to green + | S. viridochromo- 
genes green genes 
14 Cinnamomeus — Pinkish Yellowish — SS. cinnamomeus 
15. | Reticuli + | White to gray Colorless +— | S. reticulr 


118 


organisms, so far, has not been reported as 
containing any significant antibiotic-pro- 
ducing forms. Although one of the first 
preparations possessing certain antibacterial 
properties ever recorded for a culture of an 
actinomycete was said to have been obtained 
from a member of the Albus group (Gratia 
and Dath, 1925), it is open to question 
whether the particular culture was a true 
S. albus. According to Pridham and Lyons 
(1960), this organism should be considered 
as more closely related to the Griseus group. 

Species belonging to the Albus series are 
found in soil and in dust. Various early in- 
vestigators, notably Almquist, Gasperini, 
Rossi-Doria, Beijerinck, and Sanfelice, re- 
ported the isolation of organisms belonging 
to this series. 

Various systems of classification of the 
Albus series have been proposed. Attention 
may be directed here to the fact that at one 
time or another all the sporulating actino- 
mycetes, especially the saprophytic forms, 
mostly now recognized as belonging to the 
venus Streptomyces, were classified (see Bei- 
jerinck, 1900, for example) ito two groups: 
(1) A. albus (Streptothrix alba), comprising 
those forms that produce a white aerial 
mycelium and no soluble pigment; (2) A. 
chromogenus (Streptothrix chromogena), i- 
cluding those forms that produce a black 
pigment on protein media. Duché appeared 
to follow this system as late as 1934, since 
he included in his monograph (Duché, 
1934) on the actinomycetes only those spe- 
cies that were said to belong to the A. albus 
group. In view of the significance of the 
specific name ‘‘albus,” representing the type 
eulture of the genus Streptomyces, it may be 
of interest to trace the usage of this name in 
the literature on the actinomycetes. 

In presenting this historical summary, the 
writer has taken full advantage of the com- 
ments this group made by 
Baldacci (1939), who is frequently quoted 
Baldacei 


concerning 


here almost verbatim. recorded 


THE ACTINOMYCETES, Vol. II 


about 30 some of which are 
listed in Table 11. (Others were not included, 
since they are not considered as typical of 
the group). 

The name ‘‘alba”’ was first applied to an 
actinomycete culture by Rossi-Doria (1891). 
He established the characteristics of this 
organism, indicating its synonymy with the 
cultures previously characterized by Alm- 
quist (1890); he also identified it with a 
culture designated as Streptothrix Foerster, 
isolated from the air by Gasperini (1890). 
Xossi-Doria refused to accept the identi- 
fication of this organism with Streptothrix 
Foersteri Cohn. He said: ‘Nothing in 
the description given by Cohn can justify 
such an idea. In that description, in fact, 
only generic characters are given; of spe- 


synonyms, 


cific characters there does not exist even a 
shadow.’ The three cultures of Almquist 
appeared to differ little among themselves. 
Since one (culture I) was said to form a 
white crust changing in time to gray, Bal- 
dacci preferred to exclude it from the syno- 
nymity with the A. albus. Gasperini (1894) 
recognized the difference between A. albus 
and A. chromogenus. The latter possessed 
chromogenic properties, the pigment diffus- 
ing into the substrate. 

Santelice (1904) was the first to divide 
into three groups the actinomycetes now 
recognized as belonging to the genus Strepto- 
myces, using S. albus as the representative 
of the first of these groups. He noted that 
some of the cultures belonging to this group 
may produce a black pigment when grown 
on potato. He added quite significantly: 
“On the basis of this observation, a super- 
ficial observer may create a new species out 
of a pigmented culture without considering 
the fact that it originated from Str. alba.” 

Krainsky (1914) isolated from garden soil 
a culture which he described as A. albus. 
This culture produced a well developed 
growth, white at first, then becoming gray 
on certain media such as glucose agar and 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


TABLE 11 


Nature and dimen- 


pcos ene Author sions of spore 
“ 
Cladothrix dichotoma | Macé a E 


Streptothrix Foersteri | Gasperini Oval (1.0-1.5) 


Streptothrix n. 2 Almquist + 
Streptothrix n. 3 Almquist + 
Streptothrix alba Rossi- Doria | + 
Actinomyces albus Krainsky Oval (1.0) 


A, albus Waksman and 


Curtis by 1.1-1.4) 
A. albus | Jensen Rectangular (0.4-0.5 
by 2.4) 
A. albus Duché + 
A. chromogenus Gasperini Oval (1.5) 
Cladothriz odortfera Rullmann + (1.0) 
A. thermophilus Berestnew + 
A. thermophilus Gilbert + (0.5-0.6) 
A. thermodiastaticus | Bergey Oval 
A. sanninii Ciferri Round 
A. almquisti | Duché | + 
A. gougeroti Duché a 
Streptothriz gedanen- | Scheele and oo 
a | | 
sis I Petruschky 
Streptothrix candida | Petruschky + 
Streptothriz lathridii | Petruschky a 
Cladothrix invul- | Acosta and G. ao 
nerabilis Rossi 


Spherical, oval (1.2-1.6 


Comparative characters of Streptomyces albus and related species (Baldacci, 1939) 


Mycelium 


119 


Proteolytic 


action 


Chrom- 
bes ogenesis 
Substrate Aerial Gelatin | Milk 
White a 
Colorless to yellow-| White _ + 
ish 
White ~ a 
White - ~ 
Colorless to black | White ~ _ 
Colorless White (less often = + 
gray) 
White or gray Cream or gray — + a 
Cream or yellow- | White > 
ocher 
White-yellow White = ~~ 
| Ocher to black White + 
| White or chalk | Weak | + 
white 
| White 
Gray-yellow Gray ar SF 
Colorless White ar = 
Colorless White or ivory oF Weak 
white 
Yellowish - ~ 
Greenish | White } — 
Colorless | White Weak 
Colorless | White 3 te 
Colorless White + + 
| White - + 


gelatin. The aerial mycelium was produced 
readily, the medium remaining colorless. 
The spores were oval, 1 yw in size. Gela- 
tin was liquefied. Nitrate was reduced. The 
culture had no diastatic action on starch. 
Waksman and Curtis (1916) isolated from 
soil cultures of an organism considered to 
be A. albus. It was similar to that of Kvain- 
sky, although the exact identity of the two 
was doubted. The diagnosis of Krainsky 
was, therefore, amended. The aerial my- 
celium appeared either white or gray, ac- 
cording to the composition of the medium; 
the substrate growth varied from white to 
gray. The sporophores produced short and 
rare spirals. The spores were 1.2 to 1.6 by 
1.1 to 1.4 uw. The culture was nonchromo- 
genic, hydrolyzed starch, reduced nitrate, 
and liquefied gelatin. Jensen (1931) also 


isolated a culture of A. albus from soil. The 
aerial mycelium was constantly white, and 
the substrate growth cream-colored 
yellow-ochre. The culture was said to pro- 
duce cylindrical spores, 0.4 to 0.5 by 2.4 wu. 
Duché (1934) created excessively 
broad “A. albus group.’ He was severely 
criticized by Baldacci (1939), who said that 
‘df it is meant by ‘albus group’ those species 


or 


an 


that produce white aerial mycelium in cul- 
ture, they are many more in numbers than 
those described by Duché. These are also 
the ‘viridis’ species and those that show 
analogy with the ‘flavus’ forms.’? Baldacci 
further emphasized that A. albus is a well 
characterized species that does not permit 
with the ‘‘albus 
group”? of Duché comprised 18 species, of 


confusion others, while 


which 15 were new ones; among these were 


120 THE ACTINOMYCETES, Vol. II 


such forms as A. viridis, A. albidoflavus, and 
A. alboflavus, which definitely belong to 
other groups. 

In his morphological study of the actino- 
mycetes, Duché recognized five types of 
sporulation, none of which was used for 
systematic purposes. The relationship of 
aerial hyphae, spirals, and spores was not 
sufficiently emphasized. Duché stated that 
colonies may also originate from arthro- 
spores, implying thereby that any mycelial 
fragments will reproduce and multiply in 
the culture. He spoke, however, of ‘“‘mono- 
sporic”’ although he used the 
method of successive dilutions and not that 
of single-spore isolation. He documented 
the various interpretations of the species 
A. albus, stating at first that the description 
of Waksman “‘ne correspond pas tout a fait 
aux type albus de Gasperini, Rossi-Doria 
et Krainsky.”’ On comparing his own cul- 
ture with the preceding ones, he stated, 
“T’espece de Waksman and Curtis semble 
posséder toutes les propriétés de celle de 
Krainsky.... Notre espece resemble aux 
deux précédentes.”’ Baldacci concluded that 
the work of Duché, after trying to prove the 
diversity of the various interpretations in 
the literature, had not attained its purpose 
of establishing what the species A. 


colonies, 


albus 
should be. 

In proposing the genus Streptomyces in 
1943, Waksman and Henrici stated: ‘‘We 
have selected as the type species of this 
newly named genus, Streptomyces albus 
(Rossi-Doria emend. Krainsky) comb. nov. 
This species was formerly known as Actino- 
myces albus WKrainsky and first described as 
Streptothrix alba Rossi-Doria. This is one 
of the commonest and best known species 
of the group, and, although it may later be 
subdivided into further species, it 1s at 
present as definite as any other. It has been 
recently studied intensively by Duché (1954) 
and by Baldacci (1939). It is colorless, with 
ovoidal 


white aerial mycelium, forming 


spores in coiled chains on lateral branches 
of the aerial hyphae. It is proteolytic, lhque- 
fying gelatin and peptonizing milk with the 
production of an alkaline reaction in the 
latter. It does not produce any soluble pig- 
ment either on an organic or synthetic me- 
dium, but does produce a characteristic 
earthy or musty odor.” 

Pridham and Lyons (1960) have recently 
made a comprehensive analysis of the pres- 
ent status of Streptomyces albus. Their study 
was based upon a detailed examination of 
55 cultures collected from various sources. 
They came to the conclusion that ‘‘there 
has existed since about 1916, two entirely 
different concepts with regard to the nature 
of Actinomyces (Streptomyces) albus. One 
concept centers around strains with the 
following characteristics: flexuous fruiting 
bodies, colors of aerial mycelium in tints 
and shades of olive-buff (yellowish-gray or 
tan); nonchromogenicity (nability to form 
brown, deep brown, or black diffusible pig- 
ments in organic substrates); and marked 
abundance in nature” (these strains are now 
considered as comprising members of the 
Griseus group). “The other concept con- 
cerns strains that are characterized by coiled 
or spiralled fruiting bodies with catenulate 
ovoldal spores; by aerial mycelium colors 
generally interpreted as cretaceus (chalk- 
white, often with faint tinges of pink); by 
nonchromogenicity (inability to form brown, 
deep brown, or black diffusible pigments in 
organic substrata); and by their relative 
rareness in nature” (these strains are now 
considered as Albus group proper). 


Morphologic Characters 


Various methods were used in the study 
of the morphology of S. albus. Baldacci 
observed two types of mycelium. One was 
hyaline, not less than | uw in diameter, rami- 
fying more or less abundantly, and having 
an undulated appearance. The ramification 
starts perpendicularly from the point of 


SERIES AND SPECIES OF GENUS STREPTOMYCES 12] 


intersection, but it can follow in other di- 
rections or assume a Wavy appearance. This 
mycelium originates directly from the germi- 
nating spores and can be rather abundant. 
It corresponds to the first vegetative growth 
and is designated as ‘“‘substrate mycelium.” 
The second type of mycelium is more dis- 
tinctly visible than the first. It is larger in 
diameter (1.1 to 1.4 yw); it is subhyaline 
with a tendency to assume yellowish colora- 
tion. This mycelium carries abundant sporog- 
enous hyphae, scarcely ramified. The ex- 
tremities curve to hook shapes and succes- 
sively turn to spirals. This mycelium is 
white and is superimposed on the substrate 
growth; in time, it turns to dirty white or 
milky white, powdery or crusty. It is desig- 
nated as ‘‘aerial mycelium.” 

The branches of the aerial mycelium be- 
come sporophores and give rise to spores 
that are formed by contraction. The spores 
are short, oval in shape, white, and not 
without a certain polymorphism, appearing 
sometimes as short rods. One may also ob- 
serve round forms, but they must be inter- 
preted as spores seen in a vertical projection. 
The spore dimensions are 0.6 to 0.7 by 1.2 
u. According to Baldacci, the spores are 
smaller than those observed by Jensen and 
longer than those described by Drechsler. 
Baldacci was not sure, however, that Drech- 
sler’s culture corresponded to S. albus. The 
difficulty in reaching an agreement con- 
cerning the shape and measurement of the 
spores is not due, as claimed by Duché, to 
their small size, but to the time at which the 
measurements are taken and observations 
made. When the spores are united in chains 
in the sporophores, they appear longer and 
rectangular; if measured when they are 
spread in the preparation, they appear 
shorter. It is natural, therefore, that only 
the shape and dimension in the latter case 
be accepted. It should be noted that the 
free spores are not distributed on the slide 
in a uniform manner, because many remain 


attached to the glass at their smaller sur- 
face, and thus appear round. Spirals are 
abundant. 

The degeneration of the hyphae can be 
observed in the old substrate mycelium: 
protoplasm 


zones of with empty 


simulating arthrospores can be seen. This 


spaces 


phenomenon led earlier investigators, includ- 
ing Gasperini, to make unjustified generali- 
zations. This is also evident in the claims of 
Lachner-Sandoval, Vuillemin, and Grigora- 
kis, who observed this false sporulation. Al- 
though these authors did not always specify 
the particular species used in their studies, 
Baldacci was inclined to think that they 
had to do rather with a Nocardia; the vege- 
tative mycelium of the latter, when its 
growth is arrested, subdivides into frag- 
ments that look like bacillary elements. 
This type of fragmentation was studied by 
Mrskov and by Jensen. The ‘ 
irregulars”’ of Duché and others bring out 
very clearly this type of degeneration, which 
was erroneously interpreted as a type of 
sporulation. 


‘arthrospores 


Cultural Characters 


S. albus produces a colony in the form of 
a growth adherent to the substrate; it is 
wrinkled and colorless. The aerial mycelium 
appears first in the drier portion of the 
colony; it is chalk-white in color, as if lime 
had been sprayed over it. On aging, it turns 
to ivory-white; on some media, such as 
nutrient assumes a grayish tint. 
With age, growth of the culture becomes 


agar, it 


opaque or even yellowish, comparable to 
the color of the substrate, as can be ob- 
served in the cultures where the aerial my- 
celium is not produced. The formation of 
the aerial mycelium appears to correspond 
to the presence of water of condensation, 
the aeration of the cultures, and various 
other factors, independent of the strain or 
variety. The aerial mycelium is more or less 
abundant and may cover the entire colony. 


122 THE ACTINOMYCETES, Vol. II 


Concentric ring formation may be observed 
in smaller colonies, as described by Rossi- 
Doria. The cultures produce the character- 
istic moldy or soil odor. 

On synthetic agar, the initial develop- 
ment of the culture is characterized by a 
dusty or powdery white, dry growth, form- 
ing furrows or concentric rings. The sub- 
strate mycelium is formed in a thin, scarcely 
visible layer over the agar. The colony does 
not assume a vigorous aspect. The white 
aerial mycelium appears late. Pigmentation 
of the agar is seldom observed, except for 
a few strains that show feeble chromogenesis 
in this medium. 

On potato, the development is rapid, with 
small, partly confluent colonies or in the 
form of extended membranous growth that 
becomes covered with white aerial myce- 
lium. 


Biochemical Properties 


Temperature: Optimum 24-28° (24— 
44°)C, 

Gelatin: Liquefied. 

Starch: Diastatic action variable. 

Sucrose: Inverted. 

Nitrate: Reduced to nitrite. 

Antagonistic properties: The organisms 
belonging to the S. albus group are usually 
weak antagonists. Some cultures possess 


activity against gram-positive bacteria. 


Species 


KrassilInikov (1949) included 19 species 
in the Albus series. He used a combination 
of different criteria for their separation and 
identification. Tor separation of the cul- 
tures, he considered the odor produced as 
the major criterion, which is rather unrelia- 
ble. Use of this criterion is largely responsi- 
ble for the inclusion, in this group of species, 
of forms designated as aromaticus, odoratus, 
odorifer, putrificus, ete. IKrassilnikov also 
considered, for identification purposes, the 
shades of white in the aerial mycelium, tem- 


flocculus, A. 


perature relation, proteolytic and antago- 
nistic properties, the secretion of a brown 
substance, growth in acid media, production 
of ammonia and H.S, decomposition of 
rubber, and formation of coremia. He in- 
cluded in this group various thermophilic 
and thermotolerant organisms. 

Gause et al. (1957) divided the Albus 
series, on the basis of the color produced on 
a complex organic medium, into three sub- 
groups, comprising five species and one 
variety: 

a. Medium not pigmented: A. candidus, 
A. candidus var. alboroseus, and A. 
albidoflavus. 

b. Medium colored brown: A. longisporus 
and A. mirabilis. 

c. Medium 
rubidus. 

The above characterization fails to recog- 
nize some of the fundamental cultural prop- 
erties of actinomycetes, namely, the produc- 
tion of melanin pigments in protein media 
and the structure of the sporophores. The 
resulting subdivision of such a group into 


albo- 


colored brownish: A. 


subgroups thus loses all significance. Only 
one of the above species (A. albidoflavus) is 
found in’ Krassilnikov’s ‘‘albus’’ — series. 
Kutzner (1956) reported that he had ob- 
tained four strains of S. albus from different 
institutions and found them to be identical. 

Baldacci placed in the Albus series the 
following organisms, either because they 
were considered as synonyms or because 
they were believed to be closely related: 
Actinomyces albus, A. acidophilus, A. alm- 
beddardi, A. chromogenus, A. 
A. exfoliatus, A. farcinicus, A. 
gedanensis, A. gelaticus, A. 
gougeroti, A. heimii, A. kimbert, A. liesket, 
A. listeriit, A. malenconi, A. reticult, A. soma- 
liensis, A. sanninii, A. saprophyticus, A. 
thermophilus, A. wpcotti1, A. willmorec; Clad- 
othrix dichotoma, C. liquefaciens, C. imvul- 
nerabilis, C. odorifera; Oospora doriae, O. 
alpha; Streptothrix alba, Str. candida, Str. 


quisti, A. 
erythreus, 


SERIES AND SPECIES OF GENUS STREPTOMYCES 12 


dassonvillei, Str. foerstert, Str. graminearum, 
Str. leucea, Str. lathridiz, and Str. pyogenes. 
Such a large conglomeration defeats com- 
pletely the purpose of grouping, since the 
above forms vary greatly morphologically, 
culturally, and physiologically. 

A number of species can, however, be in- 
cluded in this series. It is sufficient to men- 
tion S. albus, S. calvus, and S. niveus. 

Il. Series Cinereus 
Characteristic Properties 

a. Sporophores straight or spiral-shaped. 

b. Color of aerial mycelium white to 
gray; occasionally dark humid stains or 
guttation drops. 

ce. Growth usually colorless, occasionally 
yellow to tan. 

d. Melanin-negative. 

When 
aerial mycelium, the color is characteristi- 
‘ally gray. Although it may be white at 
first, it changes to various shades of gray, 


members of this series form an 


ranging from light gray to mouse-gray to 
bluish-gray or even vinaceous-gray or black- 
ish-gray. Frequently white spots are pro- 
duced in the aerial mycelium. The substrate 
growth is often colorless or gray, occasion- 
ally becoming yellowish to buff-colored; it 
is either opaque or somewhat slimy; the 
reverse 1s usually colorless, occasionally 
turning yellow to tan. It is melanin-nega- 
tive. Occasionally a yellowish or brownish 
soluble pigment may be produced. The 
sporophores are straight, often formed in 
clusters or tufts; they may also produce 
spirals that are either open or compact. 

This series is widely distributed in nature. 
It comprises, in addition to well described 
species given here, a great number of incom- 
pletely described forms, as shown in Chapter 
i: 

A variety of antibiotics have been found 
to be produced by members of this series. 


Oo 


Some Characteristic Species 


The following species may be tentatively 
included in this series: S. craterifer, S. inter- 
medius, S. parvullus, and S. cellulosae. 

One may also include in this series various 
organisms described by Krassilnikov (1949), 
including his emendation of <A. griseus 
Kkrainsky (not S. griseus Waksman), A. 
variabilis, and A. 
This is also true of some of the forms de- 
by Gause et al. (1957) under such 


griseus griseus zonatus. 
scribed 
names as A. rubiginosus, A. griseomycini, 
A. werint, A. acrimycint, A. acrimycini var. 
atroolivaceus, and A. 


globosus, A. griseoru- 


bens. 
IIL. Series Flavus 


Characteristic Properties 


a. Sporophores long, spiral-shaped; spores 
spherical to oval. 

b. Aerial mycelium white to gray to 
mouse-gray ; color of growth yellow to golden 
yellow. 

c. Melanin-negative. 

d. Yellowish-green to golden soluble pig- 
ment may be excreted into the medium. 

e. Strongly proteolytic and antagonistic. 

The Flavus series includes a large number 
of organisms, widely distributed in nature. 
The members of the series vary greatly in 
some of their cultural, biochemical, and 
morphological properties when grown on 
artificial media. This group has been recog- 
nized 1891, 
scribed a culture under the name of Strepto- 
thrix albido-flava. Another similar culture 


since when Rossi-Doria de- 


was soon described by Gasperini (1892) as 
Actinomyces albido-flavus. Sanfelice (1904) 
designated the second of his three groups as 
Str. flava, comprising organisms isolated 
from the air. Caminiti (1907) was inclined 
to include in this group various pigmented 
forms, such as Str. cztrea and Str. chromo- 
gena. 


Numerous organisms belonging to the 


124 


Flavus series have been isolated from soil, 
dust, and other natural substrates by Krain- 
sky, Waksman and Curtis, and others. They 
have been designated by a variety of names, 
such as S. alboflavus, S. aureus, S. citreus, 
S. griseoflavus, and S. flaveolus. 

The Flavus series is characterized by 
cream-colored to yellow or golden yellow 
growth on most artificial media. The aerial 
mycelium is usually white to gray to mouse- 
gray. The sporophores are long, usually 
spiral-shaped. The spores are spherical, us- 
ually 0.7 » in diameter. No brown pigment 
is produced on protein media. A yellowish- 
green to golden pigment is often formed in 
synthetic and organic media. The various 
species in this group are strongly proteolytic 
and diastatic. Sucrose is inverted. Nitrate 
is reduced. Many of the strains are strongly 
antagonistic and are able to form active 
antibiotics, some of which have found ex- 


tensive application as chemotherapeutic 
agents. 


KrassilInikov (1949) recognized 13 distinct 
species as belonging to the Flavus series. 
Baldacci (1939), however, subdivided the 
actinomycetes with the characteristics of 
the Flavus series (various species producing 
vellow or golden growth) into a number of 
series: “aureus,” “albidoflavus,” “sulphur- 
eus,”’ “antibioticus,’ and ‘‘hygroscopicus.”’ 
Baldacci et al. (1954) included in the ‘‘Aw- 
reus”’ series such species as S. aureus, S. 
aureofaciens, S. citreus, S. fimicarius, 8. 
flavus, S. flaveolus, S. fordit, S. griseoflavus, 
S. hygroscopicus, S. microflavus, and S. par- 
DUS. 

A culture of an organism isolated by 
Takahashi (1953) in Japan was identified 
by him as S. flaveolus Waksman. To validate 
this identification, his description of this 
culture is presented in Table 12 alongside 
Waksman’s description of the original type 
culture. These data show that, in spite of 
minor variations in color characterization, 
quantitative differences in gelatin lquetfac- 


THE ACTINOMYCETES, Vol. II 


tion and nitrate reduction, and even in 
differences in antibiotic production, the 
identification of the species appears to be 
correct. 

The same is true of the characterization of 
S. parvus. The original culture of this organ- 
ism, which was used as the basis for its 
description in Bergey’s Manual, has died out 
in the collection. Benedict, of the Northern 
Regional Research Laboratory of the U.S. 
Department of Agriculture, isolated from a 
sample of soil collected in West Africa a 
culture which he identified as S. parvus. A 
comparison was made of the culture origi- 
nally isolated and described by Krainsky 
(1914), the culture isolated by Waksman 
and Curtis (1916) and reported in Bergey’s 
Manual, and the new culture of Benedict 
(Table 13). The results point definitely to 
the identity of the three cultures, thus prov- 
ing again that accurate identification of 
some species can be made by comparing 
freshly isolated cultures with written de- 
scriptions of type isolates. 

Finally, a comparison was made (Waks- 
man, 1957) of two published descriptions of 
S. flavus and S. griseoflavus, together with 
recent descriptions of two cultures that 
have been raised to the status of new species, 
namely, S. aureofaciens and S. rimosus, both 
important producers of antibiotics. The re- 
sults, presented in Table 14, show that S. 
aureofaciens and S. rimosus are sufficiently 
different from S. flavus and S. griseoflavus 
to warrant the creation of new species. S. 
aureofaciens is characterized by a deep gray 
aerial mycelium, by a lack of or limited 
spiral formation, by lmited proteolytic ac- 
tivity upon gelatin and milk, and by poor 
growth on nutrient agar. It was concluded 
that properties differentiated this 
culture sharply from the two older cultures. 


these 


S. rimosus is characterized by poor growth 
on synthetic agar and by the formation of 
abundant spirals in its aerial mycelium. 
together with certain 


These properties, 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


—_ 
~ 
oO 


TABLE 12 


Identification of Streptomyces flaveolus (Waksman, 1919; Takahashi, 1953; Waksman, 1957) 


Characteristics 


Morphology 
Sporophores 
Spores 

Synthetic agar 
Substrate growth 


Aerial mycelium 
Soluble pigment 
Calcium 
NH,Cl agar 
Substrate growth 
Aerial mycelium 
Soluble pigment 
Nutrient agar 
Substrate growth 


Aerial mycelium 
Soluble pigment 
Gelatin 
Substrate growth 
Aerial mycelium 
Soluble pigment 
Liquefaction 
Potato 
Substrate growth 
Aerial mycelium 
Color of plug 
Glucose broth 
Substrate growth 
Aerial mycelium 
Soluble pigment 
Milk 


Nitrate reduction 


malate- | 


| Cream-colored 


Waksman Takahashi 


Numerous spirals on all media 
Oval to elliptical 


Numerous spirals on synthetic media 
Spherical or oval, 0.8 by 1.2 u 


Light sulfur-yellow turning cad- 
mium-yellow 

White with ash-gray patches 

Empire-yellow 


Antimony-yellow to chamois-colored 


White, later smoke-gray 
Buff-yellow 


Pale olive-buff to yellow-ocher 
_ Vinaceous-buff to light mouse-gray 
None or faint yellowish 


Mouse-gray, with white margin 
None 


Wrinkled, white Colorless to whitish, reverse cinnamon- 
buff 
White 


| Golden yellow 


Abundant, white 
None 


Abundant yellowish pellicle Wrinkled, yellow 


White White 
Golden to faint brown Faint yellowish-brown 
| Rapid Medium 


| White 


| 


Wrinkled, cream-colored Wrinkled, golden yellow to orange 
White to seashell-pink 

Faint brown Faint brownish 
Thin, yellow pellicle 
White 

Golden 


Colonial buff to honey-yellow 

White to smoke-gray 

Yellowish (golden yellow) 

Rapid coagulation and peptoniza- 
tion 

Strong 


Rapid coagulation and strong peptoni- 
zation 
Positive 


Antibiotic production | Produces actinomycin 


other morphological and cultural differences 
between this culture and the two older cul- 
tures, justified creation of a separate species, 
especially because of the ability of S. r¢mo- 
sus to produce an important new antibiotic. 

In view of the great variability of these 
organisms and the temptation to establish 
separate species on the basis of minor differ- 
ences in pigmentation, any attempt to 
create such new species must be considered 
critically. 


Produces flaveolin 


described as SS. armillatus 
(Maney-Courtillet e¢ al., 1954) appeared, on 
the basis of the description, to be sufficiently 
close to S. rimosus to throw doubt upon its 
distinct identity. Like the latter, it produced 
spirals in its aerial mycelium; on synthetic 
agar it formed very poor growth without any 
aerial mycelium and without pigmentation; 
on nutrient agar, it produced yellow-gray 
growth with poorly developed white aerial 


A culture 


mycelium and no soluble pigment; on potato 


126 THE ACTINOMYCETES, Vol. II 
TABLE 13 
Characterization of Streptomyces parvus (Waksman, 1957) 
Characteristics | Krainsky Bergey’s 6th Edition New culture received drm N. RRL 
Substrate ; Pal Golden yellow to brick-red | Bright yellow 


growth | 


Aerial myce- | White to gray to rose-yel- 


depending on composi- 
tion of medium | 


| Poorly developed,  rose- 


Long, straight hyphae; no 
spirals 


| Short, oval 


Thin, yellow growth; thin 


lium low depending on nitro- | white; sporophores pro- 
gen source duce spirals 
Spores More or less oval, 1.6 w in | Spherical to oval, 0.9-1.3 
| size by 1.2-1.8 pu 
Synthetic | Colonies small, yellow in | Colonies small, yellow, 
agar | color, with light colored) with aerial mycelium | 
- aerial mycelium* light yellow | 
: | : | 
Nutrient Yellow growth. Aerial my- 
agar celium appears late 


white to yellow aerial my- 
celium; bright yellow sol- 
uble pigment 

Yellow growth; abundant 
white with grayish tinged 


aerial mycelium; bright 
yellow soluble pigment 
Glucose  as- | Yellow growth; white to 
paragine | gray aerial mycelium, 
agar | golden soluble pigment 
Glucose agar | Aerial mycelium light yel- Colonies small, yellow, | 
low; appears late with aerial mycelium | 


Gelatin Colonies flat or concave, 
yellow in color; gelatin | 
slowly lquefied 

Potato Colonies yellow, aerial my- | 
celium white | 

| 
| 
| 
| 
| 
Cellulose | White surface growth 


Remarks | 
| mitrate slowly; strongly | 
| proteolytic | 


| Growth good 
Produces diastase; reduces | Produces actinomycin; re- | 
duces nitrate slightly 


hight yellow 
| Colonies yellow; liquefac- 
tion medium 


Cream-colored growth drop- 
ping to bottom; good 
liquefaction; bright yel- 

low soluble pigment 
Abundant wrinkled brown- 

| ish-yellow growth; abun- 
dant sulfur-yellow aerial 
mycelium; no soluble pig- 
ment 


* Calcium malate agar 


plug, it produced yellow-gray growth with a 
faint brownish soluble pigment; on gelatin, 
it formed a surface growth with white aerial 
mycelium, with a yellowish or brownish 
soluble pigment, and good liquefaction of the 
gelatin; on milk, it produced good grayish 
erowth. These characteristics, together with 
the ability to produce oxytetracycline, defi- 
nitely placed the culture in the S. riémosus 
species. Emphasis was laid upon the fact it 


formed flat colonies, hardly folded and not 
eracked like those of S. rémosus; it showed 
concentric circles in the aerial mycelium, a 
variable property. It did not form nitrite 
from nitrate, and it did not hydrolyze starch; 
these two properties were hardly sufficient, 
however, to justify the recognition of S. 
armillatus as a new species. 

Among the various members of the Flavus 
series, the actinomycin-producers occupy an 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


TABLE 14 


Characteristics of Streptomyces flavus and allied strains (Waksman, 1957) 


Characteristics S. flavus (Bergey) S. griseoflavus (Bergey) S. aureofaciens (Duggar) S. rimosus (Sobin et al.)* 
Morphology 
Sporophores Straight, much branched, | Straight, no spirals Straight, flexuous; no spi- | Numerous spirals 
no spirals rals as a rule; occasional 
loose spirals 
Spores Oval Spherical to oval, 1.5 » | Short, cylindrical, 0.6-0.7 
long by 0.8-1.4 4 


Synthetic agar 
Substrate 
growth 
Aerial myce- 
lium 
Nutrient agar 
Substrate 
growth 
Aerial myce- 
lium 
Soluble pig- 
ment 
Gelatin 
Substrate 
growth 
Aerial myce- 
lium 
Liquefaction 
Soluble pig- 
ment 
Potato 
Substrate 
growth 
Aerial myce- 
lium 
Color of plug 


Yeast-glucose 

agar 

Substrate 
growth 

Aerial myce- 
lium 

Soluble pig- 
ment 


Milk 


Production of 
antibiotics 


Yellow to sulfur-yellow 


Straw-yellow 


Cream-colored, lichenoid 
White to light gray 


None 


Yellowish 
None 


Positive 
Faint yellowish 


Lichenoid, brownish to 
greenish-olive 
White to gray 


Brownish or none 


Rapid, lichenoid, brown- 
ish 
White, later grayish 


Yellow 


Coagulation and peptoni- 
zation 

An antibacterial 
formed 


agent 


Reddish-brown to orange 


White 


Cream-colored 

White 

None 

Cream-colored to brown- 
ish-white 

White 

Slow 


Faint yellowish 


Lichenoid, brownish to 
reddish-brown 
White to gray 


None 


Cream-colored to brown- 
ish 
White to grayish 


Yellowish 


No coagulation, rapid pep- 
tonization 


Heavy cream-colored, be- 
coming yellowish-brown. 

White, turning mouse-gray 
to brownish-gray 

Good, light brownish 


None 


None 


None 


Wrinkled, orange-yellow 


Unchanged 


Heavy, cream-colored 


White to deep gray or dark 
gray 
None 


No coagulation, no pep- 
tonization 
Chlortetracycline 


Submerged, colorless 


None 


Cream-colored to brownish 

None or white to gray- 
white 

Faint yellowish or none 

Moderate 

White 


Medium to good 


Wrinkled, ochroid 
Whitish to drab 


Yellowish-brown 
or none 


pigment 


| Good, yellowish 


Pallid drab 
Yellowish 
No peptonization 


Oxytetracycline and rimo- 
cidin 


* An earlier description of this culture on synthetic agar was incorrectly labelled; the medium was made up at that time with glu- 
cose in place of sucrose, which explains the difference between the previous and present observations. 


interesting place. A culture (No. 3491) be- 
longing to S. flavus or to S. parvus was 1s0- 
lated in our laboratories in 1948 and found 
capable of producing actinomycin. It was 
nonchromogenic and formed a straw-colored 
to yellow aerial mycelium. This culture was 
found to belong to the S. flavus subgroup; 
other cultures (Nos. 3677, 3679, and 3680) 


were included in the S. parvus subgroup. 
Another culture (No. 3686), designated as 
S. parvus, did not form any spirals. Still 
another culture (No. 3687) produced only 
limited curling of the aerial mycelium and 
might be considered an intermediate be- 
tween the two subgroups. It was suggested, 
therefore (Waksman and Gregory, 1954), 


THE ACTINOMYCETES, Vol. II 


Fiaure 31. Variation in morphology of spore-bearing hyphae in S. aureofaciens: (left) natural vari- 
ant A 377; (center) natural variant AB 374; (right) induced mutant A 377-2655 (Reproduced from: 
Backus, E. J. e¢ al. Ann. N. Y. Acad. Sci. 60: 101, 1954). 


that the whole series be designated as Flavus- 
parvus. Considerable variation was found 
among the members of this series. 


Morphological Characters 


Hyphae: (a) short, gnarled, and in clus- 
ters, with short oval spores; or (b) long, 
straight with spherical spores; or (c) long 
with long corkscrew spirals and spherical 
spores (Fig. 31). 


Physiological Characters 


Sucrose nitrate agar: Growth cream- col- 
ored, yellow to brownish to orange; reverse 
yellow to orange. Aerial mycelium cream- 
colored, straw-colored to citron-yellow, 
straw-yellow, grayish-yellow to bluish-gray 
to white, or absent. Soluble pigment hght 
yellow to brownish. 

Glucose-asparagine agar: White to cream- 
colored growth, sometimes turning orange. 
Aerial mycelium white to gray. Soluble pig- 
ment none, or brownish to yellow. 

Nutrient agar: Growth cream-colored to 
yellowish to brownish. Aerial mycelium 
white, cream-colored to 
Soluble pigment yellow to almost none. 
Cream-colored to 


eray, or absent. 


Gelatin: vellow to 


orange-yellow ring on surface. Aerial myce- 
hum cream-colored, straw-green to gray, or 
absent. Soluble pigment brownish to yellow, 
or absent. Liquefaction varies from slow to 
rapid. 

Potato: Growth abundant,  lichenoid, 
cream-colored to brownish to orange. Aerial 
mycelium white, cream-colored, gray to 
vellow. Usually no soluble pigment; occa- 
sional yellowish-orange pigment. 

Milk: Surface growth abundant or thin 
eray to black ring. Aerial mycelium white to 
gray or absent. Milk not coagulated but 
peptonized, the rapidity depending on ex- 
tent of growth. 

Antagonistic properties: Some members of 
the group produce highly important anti- 
biotics, such as the tetracyclines, that have 
found extensive application in chemotherapy 
and in food preservation (Kochi et al.,1952). 

A careful study of the literature reveals 
the fact that a large number of species found 
in nature belong to this series. Some of them 
have been well recognized and described. 
Various others may be added, but many 
have been only insufficiently described. On 
the basis of the recognized information, the 
S. flavus series may be said to include the 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


following species: S. flavus, S. flavovirens, S. 
flavogriseus, S. chrysomallus, S. celluloflavus, 
and S. viridans. 


IV. Series Ruber 


Characteristic Properties 


a. Sporophores — straight or — spiral- 
shaped. 

b. Substrate growth pink, red to red- 
orange to purple-red; pigment insoluble. 


Aerial mycelium thin, rose-white. 

ce. Melanin-negative. 

The Ruber series comprises a large, highly 
heterogeneous group of organisms. Members 
of this series have been known since 1888, 
when Macé described an organism under 
the name of Cladothrix Numerous 
other cultures under different names were 
later placed in this group. 

The Ruber series is characterized by a 
bright red, red-orange, or rose-red substrate 
growth, the color depending on the composi- 
tion of the medium and on conditions of 
cultivation. The cultures may show con- 
siderable variation in color of the substrate 
mycelium, from purple-red to light rose. The 
pigment is usually not excreted into the 


rubra. 


medium, unless the latter contains fatty sub- 
stances in which the pigment is soluble. The 
aerial mycelium is not well developed; it is 
usually produced on synthetic media as a 
thin, rose-white cover, or it is formed only 
in isolated sectors or spots. The sporophores 
are straight or spiral-shaped; the spores are 
spherical to oval, 0.7 to 0.8 by 0.8 to 1.0 wu. 

The members of the Ruber series are not 
very strongly proteolytic or diastatic. Su- 
crose is readily inverted. Some of the species 
belonging to this series are active producers 
of antibiotics. 

Baldacei did not list a Ruber series, but 
one designated as “‘roseus,’? which is close 
enough to be considered similar to it. An- 
other was designated as ‘‘melanosporeus,”’ 


129 


which the ‘‘ruber’’ series. 


Gause et al. (1957) divided the series into 


is also close to 


three subgroups on the basis of structure of 
the namely, spiral-shaped, 
straight, and tuft-forming; the last appar- 


sporophores, 


ently includes verticil-forming types. 

Some of these organisms may be con- 
sidered as forms intermediate in transition 
to the true chromogenic types. Among the 
forms closely related to this series, one may 
inciude, for example, S. melanocyclus, S. 
melanosporeus, S. melanogenes, and possibly 
also S. erythrochromogenes, S. roseochromo- 
The 


Ruber series is also related to the Fradiae 


genes, and SS. purpureochromogenes. 
and Flavus series, notably through such spe- 


cies as S. roseoflavus and possibly S. micro- 


flavus. 


Certain forms that may be considered as 
species of Nocardia are frequently included 
in this and in the next series. Sometimes even 
a new series Is created for them, as was done 
by Baldacci (1942) for ‘‘madurae.”’ 

S. albosporeus may be considered as a 
subgroup of the Ruber series. It is charac- 
terized by the formation of a rose-colored or 
red to brown substrate growth and a white 
aerial mycelium. Cultures belonging to this 
subgroup are characterized by strong pro- 
teolytic activity and by weak diastatie ac- 
tion. The sporophores are straight, with 
some close spirals. The first representatives 
of this subgroup were isolated by Krainsky 
in 1914 and by Waksman and Curtis in 1916. 

The separation of members of this series 
on the basis of carbon utilization has been 
suggested by Zahner and Ettlinger (1957), as 
shown in Table 15. 

Although a large number of species found 
in the literature may be included in the 
Ruber series, only a few have been suffi- 
ciently described. It is sufficient to mention 
S. ruber, S. niveoruber, S. albosporeus, and S. 


erythraeus. 


THE ACTINOMYCETES, Vol. II 


TABLE 15 


Utilization of carbon sources by a group of closely related 


Streptomyces species (Corbaz et al., 1957) 


2 4 = S 

Culture z z e fe » 2 z = g 3 
Sluggo Ose ee 8 Fey a ie eae 

“A a4 f io) 2S c 3 > T a = 

De Sane Ay’ 288 oR gat ee = A nt cee 

S. purpurascens a a + tr ar Seed a daiz, eee en a 
S. bobiliae SI “F ls = a + she (=) C=). [Ge 
S. cinereoruber + —- (-— = 2 = = Ve) ae as 
S. cinereoruber var. fructofermen- + (+) + (4+) + (ey ee aS ae as 


tans 


+ = good growth; (+) = 
— = no growth. 


V. Series Viridis 


Characteristic Properties 

a. Sporophores straight or spiral-shaped. 

b. Growth at first colorless, becoming 
green to dark green. Aerial mycelium white 
to gray to light green to light blue. 

ce. Melanin-negative. 

d. Soluble pigment absent or greenish. 

The species included in this series show 
considerable overlapping with the species in- 
cluded in the chromogenic series, such as S. 
viridochromogenes (syn. A. viridis (Lom- 
bardo-Pellegrino) Baldacci). 

Various other organisms that might be 
included in this series have been described. 
It is sufficient to list S. alboviridis, S. griseo- 
viridis, and S. dassonvillet. 

The following organisms may be included 
in the Viridis series: S. viridis, S. prasinus, 
S. hirsutus and S. prasinopilous. 

Several forms described by Gause ef al. 


(1957) could be ineluded in this series, 
notably A. malachiticus and A. olivaceoviri- 
dis. 


VI. Series Violaceoruber 


Characteristic Properties 
a. Sporophores produce spirals. Spores 
spherical to oval. Surface of spores smooth. 
b. Substrate growth colorless, becoming 


weak growth, questionable carbon utilization; (—) = 


very weak growth; 


red, later blue. Aerial mycelium white to 
gray with bluish tinge. 

c. Melanin-negative. Soluble red pigment 
in acid media, changing to blue in alkaline. 

A number of organisms belonging to the 
genus Streptomyces are able to produce a 
blue pigment when grown on certain media 
(Tables 16, 17). This pigment is either re- 
tained in the substrate mycelium or is readily 
dissolved in the medium; it is frequently ac- 
companied by a dark chromogenic pigment. 
The color of the pigment ranges, therefore, 
from light blue to dark blue or violet, and to 
almost black. The soluble pigment fre- 
quently changes in color with a change in 
reaction of medium, from red at an acid 
reaction to blue at an alkaline reaction. Be- 
‘ause of this change in the color of the pig- 
ment, various names, indicating the red and 
blue color combinations, have been used to 
describe the species, such as ‘‘vzolaceus,” 
“violaceoruber,” ‘‘violaceoniger,” ‘‘tricolor,”’ 
and ‘“‘pluricolor.”’ The species capable of 
producing blue pigments are divided here 
into two distinct subgroups: S. violaceorwber 
and S. vzolaceoniger. The first comprises the 
forms that produce a litmus-like pigment, 
changing from red in acid media to blue in 
alkaline; the second includes those forms 
that produce violet to dark blue to almost 
violet-black pigments on synthetic and or- 


b) 


ganic media. 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


TABLE 16 


Organism Color of aerial mycelium 


Light grayish-blue 

Grayish-yellow 

Greenish-grayish-yellow 

Bluish-gray to blue 

Greenish-brownish-gray 

Gray, sometimes with 

_ brownish tinge 

White with purple tinge 
due to substrate myce- 
lium 

Whitish-gray 


S. caeruleus 

S. coelicolor 

S. cyaneofuscatus 
S. cyaneus 

S. cyanoflavus 

S. litmocidini 


S. novaecaesareae 
(= A. violaceus 
caesart) 

. pluricolor 


—R 


S. tricolor 
S. violaceoruber 


Light brown to light gray 
Ash-gray 


| Ash-gray 
Mouse-gray 


S. olivaceus 
Streptomyces sp. No. 
169 


—; seldom 


Streptomyces species, producing a blue pigment (Kutzner and Waksman, 1959) 


Melanin 


pigment Author 


Spirals Spore surface 


= — Baldacci, 1944 

_ Smooth Miller, 1908 
Gause et al., 1957 
Krassilnikoy, 1949 
Funaki et al., 1958 
Gause et al., 1957 


+ Spiny 


+++ 1 


atts 
+ = Waksman and Cur- 
+ | | tis, 1916; Waks- 
| man, 1919 
ok Berestnew, 1897; 
| Krassilnikovy, 
| 1949 
Wollenweber, 1920 
Waksman and Cur- 
tis, 1916; Waks- 
man, 1919 
Corbaz et al., 1957 
oS Kurosawa, 1951 


++ 


Smooth _ 


— Smooth = 


The first organism belonging to this series 
was isolated, in 1891, by Rossi-Doria and 
described as Streptothrix violacea. It was later 
studied by Gasperini (1894), and by San- 
felice (1904) as one of the three important 
constituent groups of actinomycetes. 

Baldacci (1942) designated the series as 
“wolaceus,” which he did not differentiate, 
however, from the subgroup designated here 
as violaceoniger. Gause et al. (1957) created 
a new series, ‘‘roseoviolaceus,”’ which logically 
belongs in this series; they also included in 
their series “‘violaceus’? a variety of other 
forms that logically belong to this series. 
Ettlinger et al. (1958) designated as azureus 
the light blue pigmented forms. 

This series is not known for the production 
of any important antibiotics, although coeli- 
colorin has been reported for cultures of S. 
violaceoruber and chartreusin for S. char- 
treusts. 

The following species may be included in 
this series: S. violaceoruber, S. novaecaesareae, 
S. cyanofuscatus, and S. litmocidini. 


Some of the melanin-producing forms, 
such as S. violaceochromogenes, may also be 
included in this series. Many of the forms 
described by Gause et al. (1957) also belong 
here. These include A. coerulescens, A. glau- 
cescens, A. bicolor, A. 
coeruleofuscus, A. violaceorectus, A. 
color, A. litmocidini, A. 
griseoruber, A. 


coeruleorubidus, <A. 
prani- 
viridoviolaceus, A. 
griseorubiginosus, A. cinna- 


barinus, and others. 
VII. Series Fradiae 


Characteristic Properties 


a. Sporophores usually straight; occasional 
loops and spirals. 

b. Substrate growth yellow-orange to or- 
ange. Aerial mycelium seashell-pink, espec- 
ially on potato agar and on glucose-aspara- 
gine agar. 

c. Melanin-negative. 

This represents a fairly large group of or- 
ganisms, widely distributed in nature. S. 
fradiae was first isolated and described by 


TABLE 


THE ACTINOMYCETES, Vol. II 


17 


Blue-pigmented substances produced by actinomycetes (Kutzner and Waksman, 1959) 


Solubility Author 


| 
Preparation Organism | Melting point | 
| | 
| 
a? a = | : i 
| [c 
Amylocyanin S. coelicolor | | 
| 
Litmocidin | Nocardia cyanea 144-146 
| 
| 
| 
‘oelicolorin S. coelicolor | 142-146 
| 
| 
Cyanomycin | S. cyanoflavus 128 
| 
Granatacin S. olivaceus | 204-206 
| 
| 
Actinorhodin S. coelicolor 270 
| \ 
(decomp) 


290-300 
(decomp) 


Streptocyanin Streptomyces sp. | 


Anthoecyanin _S. violaceoruber 


Anthocyanin S. coelicolor 


Anthocyanin S. violaceoruber 


Hydroactino- | Streptomyces — sp., 
chrome producing violet | 
growth and pig- | 
ment | | 
Lipoactino- | 
chrome | | 


Waksman and Curtis (1916). Of the two 
neomycin-producing cultures isolated by 
Waksman and Lechevalier in 1949, one 
formed no spirals and thus agreed with the 
original description of the organism; the sec- 
ond produced some spirals of the closed type. 
Differences were also observed in the shade 


| Very soluble in acetone, ethyl- 


| Soluble in water 


Inwaterandindimethylforma- | Miiller, 1908 
mide; insoluble in other sol- 
vents | 
Shghtly soluble in water at an | Gause, 1946; 
acid reaction and extracted | Brazhnikova, 
from it by ethanol, ether, or | 1946 
amylacetate 
Kominami, 1949; 
acetate, or chloroform; solu- Hatsuta, 1949 
ble in ethanol, methanol, 
benzene, or ether; insoluble | 
in petroleum ether 
Extracted from water at 
alkaline reaction by chloro- | 
form or methylenechloride 
Extracted from water at an 
acid reaction by acetone; | 
soluble in ethylacetate, and | 
dimethylsulfoxide; insoluble | 
in petroleum ether 
Soluble in pyridine, piperi- 
dine, or phenol; weakly sol- 
uble in dioxane or acetone; 
insoluble in ether, CS: , 
CCl, or petroleum ether | 
Soluble in acetone, dioxane, or | Tonolo 
pyridine | 1954 
Extracted with hot cold | Kriss, 1936 
water and dilute alcohol 
| Kriss, 1937 


Funaki et al., 
1958 


an | 


Corbaz et al., 


1957 


Brockmann et al., 
1947, 1950, 1955 


Ci Males 


or 


Extracted with hot or cold | 
water and dilute alcohol | 
| Frampton and 
| Taylor, 1938 
Kriss, 19386 


| 


I 


Insoluble in water 


of color of the aerial mycelium on synthetic 
media. 

Some of the strains of S. fradiae were 
found capable of producing certain antibi- 
otics, notably members of the neomycin com- 
plex, as well as the antifungal agent fradicin. 
Several other species reported in the litera- 


SERIES AND SPECIES OF 


ture appear to be related to the Fradiae 
series. Baldacci et al. (1953) at first did not 
recognize this organism as representing a 
distinct series, and apparently considered it 
as a within the 
Later, Baldacci 
(1956) gave it series characteristics. 

Gause et al. (1957) divided the Fradiae 


member “roseus”’ series. 


however, and Comaschi 


series, on the basis of spiral formation, into 
two subgroups: one, spiral-forming, com- 
prising A. roseoflavus; the other, nonspiral- 
forming, comprising A. fradiae proper; other 
species and varieties were included in both 
subgroups. Most of the members of a new 
series, designated as ‘‘fuscus,’’? could also be 
considered as members of the /’radzae series. 

Waksman and Scotti (1958) divided the 
Fradiae series into three subgroups. These 
were described briefly as follows: 

I. Substrate growth on synthetic media 
thin, smooth, colorless, almost entirely lim- 
ited to the surface of the medium; occasion- 
ally colored orange-yellow. Aerial mycelium 
light pink, seashell-pink, or salmon-colored. 
Some strains produced little if any growth 
on synthetic media. Best sporulation took 
place on potato agar and on glucose-aspara- 
gine agar. On organic media, growth was 
smooth to wrinkled, yellowish or orange- 
vellow to orange-brown,; aerial mycelium, if 
present, was white to seashell-pink. On cer- 
tain media, a soluble, pink to salmon-colored 
pigment was produced. Morphologically, all 
strains formed a straight aerial mycelium; 
some cultures, however, were able to form 
hooks and loops, and even occasional spirals, 
on certain media. These strains were con- 
sidered as representing typical S. fradzae 
proper. 

Ila. On synthetic media, substrate growth 
thin, colorless, limited almost entirely to the 
surface of the medium; aerial mycelium 
white. On organic media, growth cream- 
colored to yellowish; aerial mycelium thin, 
white to grayish-white. On yeast-glucose 
agar, growth orange to brownish to greenish; 


GENUS STREPTOMYCSE 133 


aerial mycelium white. Abundant spirals 
were found in the aerial mycelium. 

IIb. On synthetic media, growth very 
poor. On organic media, growth generally 
poor; growth best on yeast-glucose agar. 
No aerial mycelium was formed. 

A detailed characterization of subgroup I 
is given in Table 18. Among the other species 
apparently closely related to this section is 
S.  kanamyceticus (Okami and Ume- 
ZAWAa). 

The following species may be included in 
the Fradiae series: S. fradiae, S. luridus, S. 
albosporeus, perhaps also S. roseus and S. 


fuscus. 


VILL. Series Griseus 


Characteristic Properties 

a. Sporophores straight, produced in tufts. 
Spores oval; surface smooth. 

b. Growth colorless to olive-buff. Aerial 
mycelium water-green to grass-green to gray. 

c. Melanin-negative. 

d. Strong proteolytic activities. Produce a 
variety of antibiotics. 

An organism, under the name of A. griseus, 
was first isolated and described by Krainsky 
in 1914. Its substrate growth on artificial 
media was colorless; only a small amount of 
yellowish soluble pigment was produced. The 
aerial mycelium was of a green-gray color 
on both organic and synthetic media. When 
the concentration of nitrogen in the medium 
was increased to 0.005 per cent, the aerial 
mycehum became white. The culture was 
only weakly proteolytic. 

Soon afterward, in 1915, Waksman and 
Curtis isolated several cultures of what ap- 
peared to be the same organism, the com- 
parison with Krainsky’s description being 
based primarily on the color of the aerial 
mycelium. Since this work was done during 
the of World War I, Krainsky’s 
original strain could not be obtained for com- 


years 


parative studies. The new culture was des- 


THE ACTINOMYCETES, Vol. II 


154 


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SERIES AND SPECIES OF 


ignated as A. griseus Krainsky, although 
certain marked differences were observed 
between the two isolates. 

Since no type culture of Krainsky’s organ- 
ism was available for comparison to any 
investigator, all the subsequent descriptions 
were based upon the Waksman and Curtis 
culture, which was distributed to all collec- 
tions in the world. 

In 1919, Waksman amended the descrip- 
tion of Krainsky, as follows: ‘‘This organism 
was isolated numerous times from the soil. 
The name A. griseus was used before by 
Krainsky so that the description of the latter 
is itself an amendment. Although this or- 
ganism was originally identified with the 
organism described by Krainsky under the 
same name (from description only, without 
any actual comparison of cultures), this 
identification should be, therefore, corrected. 
The culture described here possesses a very 
strong proteolytic power, while Krainsky 
stated that his culture was not strong pro- 
teolytically.”’ 

The differences between the two cultures 
can be briefly summarized as follows: A. 
griseus Krainsky produced a greenish-gray 
to dark gray aerial mycelium, with a green- 
ish-yellow soluble pigment in older cultures; 
growth on potato was grayish, with white- 
gray aerial mycelium; Krainsky never stud- 
ied the morphology of his organism, except 
for the shape (oval) and size of the spores. 
A. griseus Waksman and Curtis produced a 
water-green to yellowish-green aerial myce- 
lium; the sporophores were straight and were 
formed in tuft-like masses; growth on potato 
was yellowish, wrinkled, and without any 
soluble pigment. 

Tn spite of these differences, Waksman 
hesitated at first to change the name of the 
culture which he and Curtis first isolated. 
This hesitation was due partly to the faci 
that the organism was found to undergo 
considerable variation upon continued cul- 
tivation on artificial media. The substrate. 


GENUS STREPTOMYCES 


the temperature of incubation, the length of 
the incubation period, the amount and na- 
ture of inoculum, all tended to exert an in- 
fluence upon the morphological and cultural 
characteristics of the organism. At one time 
milk was clotted at 37°C in 2 days and then 
peptonized; at another time, under the same 
conditions, clotting of the milk required 5 
to 6 days; at still another time, the milk 
in some tubes was not clotted at all but was 
rapidly peptonized. There were other recog- 
nizable changes or Drechsler, 
studying the morphology of the Waksman 
and Curtis culture, found that the aerial 
mycelium showed proliferation of fertile 
branches at moderately close intervals along 
the axial hyphae, thus suggesting tuft for- 
mation. This phenomenon alone would have 
definitely indicated that the culture should 
have been identified as a distinct species. 
In August 1943, in the laboratories of the 
Department of Microbiology of the New 
Jersey Agricultural Experiment Station, a 
culture was isolated which produced the 
highly important antibiotic designated as 
streptomycin. Upon careful examination, 
this culture was found to be similar to the 
Actinomyces griseus described by Waksman 
and Curtis in 1916. Since, in the meantime, 
Waksman and Henrici had proposed that the 
generic name for the sporulating forms of 


variations. 


actinomycetes be changed from Actinomyces 
to Streptomyces, the organism was named 
Streptomyces griseus. This name has been 
universally recognized, since 1944, as the 
official one for the streptomycin-producing 
organism, and has been so designated in nu- 
merous throughout the 
world. A detailed description of this species 
was published in 1948 (Waksman, Reilly 
and Harris). 

Baldacci et al. (1954) subjected the Griseus 
series to a detailed study. They recognized 


other collections 


that this representative species had come to 
the fore as a result of the important role 
that it played in the production of antibi- 


156 


otics. They emphasized that although first 
listed by Krainsky in 1914, S. griseus was 
amended and described in detail by Waks- 
man in 1919. They further added: 

“Tf we examine the characteristics given 
by Krainsky we are led to link this species 
with A. viridis. This conclusion appears still 
more logical when we study the coloured 
tables prepared by Krainsky. However, in 
view of the impossibility of comparing 
Kkrainsky’s original strain and the difficulties 
that would arise if one did not accept Waks- 
man’s amendment for a species so generally 
studied in laboratories, 1t appears advisable 
to take as definite the characteristics speci- 
fied by the American author and given in 
Bergey’s Manual .... Numerous strains have 
been isolated by us and compared, with 
satisfactory results, with Waksman’s strains. 
_..There is a considerable body of literature 
dealing with this species which has a faculty 
for mutation.” 

Baldacci and Comaschi later (1956) wrote: 

“The examination of Krainsky’s descrip- 
tion and colored pictures would suggest that 
this species belongs to A. virzdis Lombardo 
Pellegrino (1903). The comparison between 
Krainsky’s and Waksman’s descriptions 
gives evidence—as even Waksman has par- 
tially pointed out—to the difference of 
proteolytic activity and. according to our 
opinion, the very important difference of the 
color of the sporulating colonies which are 
greenish in Krainsky’s description. If we ac- 
cept Waksman’s correction of the species 
and compare his descriptions with our 
strains, we find a perfect identity. Since it is 
impossible to compare the original strain of 
Krainsky with the others, the acceptance of 
the correction proposed by Waksman offers 
the advantage of maintaining the name 
“griseus” for an actinomycete so largely 
spread out and studied in laboratories, so 
that we agree with it according to this mean- 
ing.” 


Many other cultures of S. griseus have 


THE ACTINOMYCETES, Vol. II 


since been isolated from soils, river muds, 
animal excreta, water, dust, and other nat- 
ural substrates. Not all of them were found 
capable, however, of producing streptomy- 
cin; the majority of these cultures were 
either inactive or produced other antibiotics, 
such as cycloheximide, grisein, streptocin, 
actinomycin, and candicidin. Some of the 
cultures yielded a mixture of streptomycin 
with other antibiotics. The ability to form 
streptomycin was at first considered as a 
strain, rather than a species, characteristic; 
later, however, it was decided (Waksman, 
1959) to raise S. griseus to the status of a 
the — streptomycin-producing 
strains to a species status, Streptomyces 
griseus, Waksman and Henrici. 

Several procedures were developed for the 
isolation from natural substrates and for the 
identification — of 
strains of S. griseus. These methods were 
based on certain physiological properties of 
the organisms and on the nature and ac- 


series and 


streptomycin-producing 


tivities of the streptomycin formed by them: 

1. Tolerance to fairly high concentrations 
of streptomycin in the medium. When a 
soil or other natural material was plated 
out on a medium containing 50 mg of strep- 
tomycin per liter, the great majority of bac- 
teria and actinomycetes failed to develop on 
the plate. Most of the actinomycete colonies 
were found to be of the S. griseus type. 

2. Ability of certain resistant strains of 
test bacteria to grow in the presence of 
streptomycin. 

3. Sensitivity to a specific actinophage. 
When cultures of S. griseus are tested for 
their sensitivity to a specific actinophage 
which is active only upon the streptomycin- 
producing species, the inactive forms or those 
producing other antibiotics can be easily 
eliminated. 

4. Utilization of streptomycin-dependent 
strains of bacteria in testing for strep- 
tomycin. When a culture of S. griseus or of 
another organism suspected of producing 


SERIES AND SPECIES OF GENUS STREPTOMYCES 137 


streptomycin was finally selected and grown 
in a liquid medium, the streptomycin-like 
nature of the antibiotic could be established 
by adding the culture filtrate to a nutrient 
broth and inoculating the latter with a strep- 
tomycin-dependent strain of Escherichia coli 
or of some other bacterium. Growth of the 
bacterium definitely established the fact that 
the unknown antibiotic was streptomycin. 

5. Cross-streaking the unknown cultures 
on a suitable agar medium toward known 
streptomycin-producing cultures. The latter 
exerted only a shght inhibiting effect upon 
the unknown streptomycin-producers. 

Usually some soil or other material is 
plated on ordinary agar media favorable to 
the development of actinomycetes; colonies 
were picked and tested. The S. griseus col- 
onies could easily be recognized by the pale 
green to grayish-green shade of their aerial 
mycelium. A suitable agar medium can also 
be seeded with living cells of a nonpatho- 
genic strain of Mycobacterium tuberculosis 
and various dilutions of soil used for plating 
purposes. The plates are first incubated at 
28-30°C for 2 or 3 days, to enable the acti- 
nomycetes to develop. This is followed by 
further incubation of the plates at 37°C for 
the development of the test bacterium. Col- 
onies that have the capacity of inhibiting 
growth of the bacterium are found to be 
surrounded by clear zones. 

The antibiotic potency of an active culture 
of SS. griseus was found to be fairly constant, 
in spite of the ability of the culture to give 
inactive variants. Highly active 
strains tend to retain their relatively superior 
streptomycin-producing potency, 
poor strains usually remain weak producers 
of this antibiotic. For the commercial pro- 
duction of streptomycin, however, it is es- 
sential to select continuously the most ac- 


rise to 


whereas 


tive strains. 

Since the streptomycin-producing culture 
isolated in 1943 was found to be identical 
with the one described by Waksman and 


Curtis, 1t must be considered in the light of 
that description. The same is true of the S. 
griseus strains isolated later and found to 
be able to produce grisein, candicidin, vio- 
mycin, and actinomycin. There are certain 
differences in the cultural and biochemical 
properties of the various strains belonging 
to the Griseus series, especially in their ability 
to produce various antibiotics and in their 
sensitivity to different phages. This justifies 
the separation of the group into several dis- 
tinct species. 

The morphological and cultural properties 
of certain cultures belonging to the Griseus 
series are given in Table 19. 


Characterization 


The Griseus series is characterized by cer- 
tain morphological and cultural properties 
that make possible its identification and 
ready distinction from other groups belong- 
ing to the genus Streptomyces. As more and 
more cultures of S. griseus were isolated, it 
became recognized that this is a large series 
of organisms, the members of which vary 
greatly in their physiological properties and 
in their ability to produce various antibiot- 
ics. 

Waksman and Curtis described S. griseus 
as producing on sucrose nitrate agar a thin, 
spreading growth, developing deep into the 
medium, at first colorless, then turning olive- 
buff. This color may be lost on successive 
transfers. The aerial mycelium is thick, pow- 
dery, water-green in color. No soluble pig- 
ment was observed; the reverse of the growth 
became brownish in 24 days. On gelatin, 
at 18°C, S. griseus produced a greenish-yel- 
low or cream-colored growth developing deep 
into the substrate; the aerial mycelium was 
white-gray with a greenish tinge. There was 
no soluble pigment; liquefaction of the gela- 
tin was rapid. The culture was capable of 
utilizing a variety of carbohydrates, includ- 
ing pentoses, hexoses, sugar alcohols, and 
organic acids. It was also able to obtain its 


138 


THE ACTINOMYCETES, Vol. II 


TABLE 19 


Morphological and physiological properties of certain strains and one mutant of Streptomyces griseus 


| Synthetic agar | Glycerol agar Gelatin* Potato | Anebi 
ea): | | 2 
Se An eENO aL yeep ar TG | Sea eee: 
Growth oe celta | Growth eyes Growth aves ties 
—— —|—— = i— - | | 
1915 W and C | Sporophores Cream-col- | Powdery, | Cream-col- White to | Cream-col- White to | Of 
(N. J. strain) long, formed ored water- | ored | greenish- ored or greenish- | 
in tufts, no | green yellow greenish- yellow 
spirals color yellow 
1915 W and C Sporophores Cream-col- Powdery, | Cream-col- White to | Greenish- Grayish ++ 
(Holland long, formed ored, turn- water- ored greenish- | yellowor | 
strain) in tufts, no ing olive- green yellow cream - | 
spirals buff color | | colored | 
1943 Strepto- Tufts, no Cream-col- Powdery, Cream to Cream-col- | Greenish | Greenish |-++-+-+ 
mycin pro- spirals ored, turning water- olive- ored yellow or | tinge 
ducer olive-buff green green cream- 
color colored | 
Grisein pro- Tufts, no Cream_-col- | Light gray Cream-col- Cream-col- | Greenish- | Grayish } ++ 
ducer spirals ored to green- ored ored to yellow 
ish | greenish | 
Rhodomycetin | Tufts, no | Vinaceous White to | Carmine- Gray +++ | Gray + 
producer spirals gray red | | | 
| | 


* Brownish pigment produced by some strains. 
+ Mutant of this culture produced an antibiotic. 


nitrogen from a variety of compounds, in- 
cluding both inorganic and organic forms. 

In studies of streptomycin-producing 
strains, Carvajal (1946) characterized the 
morphology and life cycle of S. griseus in 
greater detail. The substrate mycelium when 
young is well branched, typically in a mon- 
opodial form. Transverse septa are formed 
in virtually all cases in the delimitation of 
the reproductive cells. Reproduction occurs 
by means of unicellular asexual’spores and 
conidia, which are exogenously borne in 
chains on the aerial mycelium. The spores 
are of various shapes: barrel, oval, bean, 
spherical, and cylindrical. Differences in 
shape and size are found often, even 
among the spores of the same chain. Mature 
aerial spores often show small fragments of 
transparent film adhering to the outside wall. 
The spores germinate at one end or at both 
ends, usually from the points at which they 
are attached to the adjacent spores or to the 
hypha. Hyphal fusions and germ tube fusions 
also can be observed. Carvajal reported that 
he had demonstrated a nucleus in the germ 
tubes of S. griseus in the young mycelium 


and in the developing spores. The nuclei 
were said to be well distributed throughout 
the cytoplasm of the mycelium; the spores 
may be uninucleate or multinucleate. 
Gottlieb and Anderson (1947) studied the 
course of spore germination and of develop- 
ment of the mycelium in submerged cultures 
of S. griseus. The exact time of spore germi- 
nation was difficult to determine, only an 
elongation of the spores being observed. 
After 6 hours, the mycelium was found to 
consist of some small hyphae and of longer 
branched hyphae which tended to develop 
into masses of mycelium consisting of a 
dense solid center and a_ periphery of 
branched radiating hyphae. Within 24 to 
30 hours, the entire body of the medium 
was filled with these mycelial clumps. The 
culture appeared viscous at this stage. After 
48 hours, the mycelium began to fragment, 
and spores were produced. At 84 hours, det- 
inite lysis of the mycelium took place; the 
dense central core of the masses of growth 
disintegrated into granular pieces. 
Measurement of viscosity and weight of 
mycelium revealed an increase which reached 


SERIES AND SPECIES OF 


a maximum at 24 to 30 hours, followed by 
a decrease up to about 96 hours; a gradual 
leveling of growth then took place. 

Growth of S. griseus in stationary cultures 
reaches a maximum in 10 days, whereas 
maximum growth in submerged cultures is 
usually attained in 3 to 5 days. This is fol- 
lowed by lysis of the mycelium. Growth of 
the organism is accompanied by a gradual 
rise in pH value of the culture and in the 
ammonia and amino nitrogen content; the 
total nitrogen in the mycelium tends to be 
higher during the active stages of growth. 
The production and accumulation of strep- 
tomycin parallel the growth of the organism, 
reaching a maximum when lysis just sets in; 
this is followed by a decrease when the rate 
of lysis reaches a maximum. 


Metabolism 


The metabolic changes of S. griseus in a 
elucose-peptone-meat extract medium have 
been found by Dulaney and Perlman (1947) 
to fall into two phases. During the first 
phase, the organism grows rapidly and forms 
extensive mycelium; this is accompanied by 
a reduction in the quantity of soluble con- 
stituents in the medium, namely, the nitro- 
gen, the inorganic phosphate, and the avail- 
able carbohydrate; the quantity of lactic 
acid present is first increased and then uti- 
lized to some extent; the oxygen demand is 
high, and the Qo. values may reach 150; 
little streptomycin is produced; the soluble 
‘carbon content of the medium during the 
growth phase rapidly falls as the glucose is 
utilized; about 50 per cent of the carbon 
appears to be unavailable to the organism 
during the first stage; the nitrogen content 
of the mycelium varies with age. During the 
second or autolytic phase of growth con- 
siderable lysis sets in; streptomycin is pro- 
duced actively, and the pH of the medium 
rises; the quantity of mycelium is decreased 
as a result of lysis; the lactic acid content 
remains more or less constant, as does the 


GENUS STREPTOMYCES 139 


soluble carbon content of the medium; the 
oxygen demand slowly decreases; the am- 
monia nitrogen, soluble nitrogen, and inor- 
ganic phosphate contents of the medium 
rise rather markedly, paralleling the autoly- 
sis of the cells. 

Ammonium compounds, but not nitrates 
are favorable sources of nitrogen for growth 
and streptomycin production. S. griseus 
rapidly assimilates phosphate in a phos- 
phorus-poor medium. An excess of phos- 
phorus has a depressive effect both upon 
growth of the organism and upon strepto- 
mycin production. 

The supplementary addition of amino ac- 
ids or of more complex organic compounds 
has been found to stimulate production of 
streptomycin. Kiser and McFarlane (1948) 
found that, of the amino acids, histidine is 
essential for both mycelial growth and strep- 
tomycin production; inositol also increased 
the yield of both; valine favored the latter, 
and aspartic or glutamic acid the former. 
If the salt concentration is low, most of the 
streptomycin will be found in the mycelium, 
thus suggesting that streptomycin is a prod- 
uct of intracellular synthesis. Woodruff and 
Ruger (1948) reported that yields as high as 
1 g of streptomycin per liter are produced 
by S. griseus in media containing proline 
as the only source of nitrogen. 

The ability of S. griseus to form an en- 
zyme (mannosidostreptomycinase) which de- 
composes mannosidostreptomycin into strep- 
tomycin and mannose has been recently 
demonstrated. This enzyme is not produced 
by other actinomycetes or fungi (Volume I, 


p18): 
On a dry basis, the mycelium of S. griseus 
contains about 16 per cent ether-soluble 


material and about 37 per cent cold water- 
soluble substances. Little study has been 
made of the specific chemical composition 
of these and other fractions. 

Stokes and Gunness (1946) grew S. griseus 
in stationary cultures in a nutrient medium 


140 


containing 0.5 per cent meat extract and 1 
per cent glucose. The cell material was dried 
and then hydrolyzed by acid or alkali. The 
amino acid composition of this material, on 
a percentage basis of the dry material, was 
as follows: total nitrogen, 9.14; histidine, 
0.84; arginine, 2.90; lysine, 2.13; leucine, 
3.73; isoleucine, 1.49; valine, 3.40; methio- 


2.33; phenylalanine, 


nine, 0.55; threonine, 2.33; 
1.67; tryptophan, 0.62. 

In addition to the two forms of strepto- 
mycin, S. griseus produces several other 
antibiotics. Ether extracts from the myce- 
lium of the organism yield a substance 
designated as streptocin, which is active 
against bacteria but not 
against gram-negative forms. Another anti- 
biotic, designated as cycloheximide, can be 
isolated by extracting the crude submerged 
eulture with chloroform, evaporating the 
extract, and dissolving the residue in meth- 
anol. Cycloheximide is not active against 
bacteria but has strong antifungal prop- 
erties; it is particularly active against yeasts, 


gram-positive 


Varieties and Mutants 


The Griseus series represents a large? 
widely distributed, and variable 
organisms. It has long been recognized 
(Waksman, 1959) that this series should be 
divided into several species. The formation 
of different antibiotics by the various species 
offers an excellent supplementary basis for 
such subdivision. The many cultures isolated 
and studied in detail can thus be classified 
into five distinct species. 

1. Streptomyces 


group. ot 


Waksman and 
Henrici. This comprises strains of S. griseus 
which produce streptomycin; they also pro- 
duce cycloheximide. 
2. Streptomyces 


QriSeUs 


Waksman. 


Strains of S. griseus which produce grisein or 


griseinus 


erisein-like substances. These strains are as a 
rule resistant to actinophage. Benedict and 
Lindenfelser (1951) demonstrated that a 


majority of streptomycin-producing strains 


THE ACTINOMYCETES, Vol. II 


of S. griseus form a green soluble pigment 
in calcium malate medium and a_ yellow 
pigment in calcium succinate medium; on 
the other hand, the grisein-producing strains 
of this organism do not form any green or 
yellow pigments in these media, although 
they show the typical greenish pigmentation 
of the aerial mycelium. 

3. Streptomyces coelicolor (Miller) emend. 
Kutzner and Waksman. This species com- 
prises strains which produce the antifungal 
agent candicidin but no antibacterial sub- 
stance. The first organsim belonging to this 
species was Isolated by Miiller (1908), and 
designated Streptothrix coelicolor. It produced 
a blue pigment similar to that formed by a 
diphtheroid organism which he called Bacil- 
lus coelicolor. The culture was a_ typical 
Streptomyces and formed concentric rings in 
its aerial mycelium. It developed well at 
room temperature and at 36°C. It grew on 
gelatin, with gradual liquefaction but with- 
out pigmentation. On agar media containing 
5 to 10 per cent dextrin, but not in glycerol 
media, a brown pigment was formed. The 
culture formed no aerial mycelium on ordi- 
nary agar media, unless serum, glycogen, 
dextrin, or starch was added. When glucose, 
sucrose, arabinose, or other sugars were used, 
no aerial mycelium was formed. 

Miller emphasized two important activ- 
ities of S. coelicolor: It possessed antagonistic 
properties, and it was active against Ozdium 
lactis. Miller was thus one of the first to 
demonstrate activities that were to make the 
whole group of actinomycetes famous. Miil- 
ler also studied the pigment extensively; he 
called it amylocyanin; it is produced best on 
potato media when grown at 30°C, but not 
at 36°C. 

4. Streptomyces californicus Waksman and 
Curtis. This species comprises strains which 
produce viomycin, active against gram-posi- 
tive bacteria only. Burkholder et al. (1955) 
classified these organisms as strains of S. 
Waksman (1958) 


griseus var. purpureus. 


TT 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


TABLE 20 


141 


Comparison of cultural characteristics of four strains of Streptomyces californicus with Streptomyces griseus on five different media 


(Burkholder et al, 1955) 


Agar medium | Mycelium S. floridae S. californicus S. puniceus S. vinaceus S. griseus 3475 
Glycerol as- | Substrate | White to light White to light Purple White to light White to light yel- 
paragine | gray to slight | gray to purple gray to slight low 
purple purple 
Aerial White White White to light White to light White to light 
gray-green gray-green gray-green 
Glucose tryp- | Substrate | Black with slight| Gray to slight | Gray to black Gray to black Light gray-yellow 
tone purple tinge black 
Aerial White to slight Slight growth, | Light gray-pink Light gray-pink White to gray-pink 
| gray-pink | white to light gray- | toslight light 
green | gray-green 
Starch syn- Substrate | Purple Light gray to slight! Purple White tolightgray | Light yellow-gray 
thetic | purple to slight purple 
| Aerial White to light | White White to light | Light gray-green | Light gray-green 
gray-green gray-green 
Calcium malate) Substrate | White to light White to light | White | White | Gray-yellow to 
yellow yellow | slight light 
| | brown 
| Aerial White White | Light gray-green | White White to slight 
| gray-green 
Nutrient | Substrate | White to slight White to slight | White to light | White to light Light gray-yellow 
light yellow light yellow yellow | gray-yellow 
Aerial White | White White White to slight White to slight 
| light gray-green| gray-green 


suggested that they be raised to the status 
of a species within the S. griseus section. A 
further study of this species points to its 
identity to S. californicus, which has _pri- 
ority in species designation. 

5. Streptomyces chrysomallus (Lindenbein) 
Waksman. These comprise strains which 
produce actinomycin. Welsch et al. (1957) 
studied 51 cultures of Streptomyces for their 
susceptibility to seven actinophages; 43 
of the strains produced actinomycin and 
eight represented nonactinomycin-producing 
strains of S. griseus. Certain actinomycin- 
producing organisms, including the Linden- 
bein culture of S. chrysomallus and a culture 
of S. parvus, were considered to belong to 
the S. griseus section. A detailed study of the 
actinomycin-producing organisms has _ re- 
cently been made by Solovieva and Die- 
lova (1960). 

Various other organisms belonging to the 
S. griseus section are able to form at least two 
other antibiotics. One of these, cyclohex- 
imide, is active only against fungi, and 
another, streptocin, is active against certain 


protozoan-like organisms. No detailed study 
has as yet been made of these strains in an 
effort to raise them to species status. 

There are also those strains of S. griseus 
that produce no antibiotic at all, at least as 
far as one is able to detect by available 
methods. 

The streptomycin-producing strains of S. 
griseus give rise readily to mutants. Two 
such mutants have been reported: One was 
a colorless form, producing no aerial myce- 
lium, forming no streptomycin, and sensitive 
to this antibiotic; Dulaney et al. (1949) re- 
ported, however, on a colorless mutant that 
produced streptomycin. The other was a 
pigmented mutant, forming pink to vina- 
ceous-colored substrate growth and an aerial 
mycelium typical of S. griseus; this mutant 
formed no streptomycin but gave rise to 
another 
was not 
teria. According to Kutzner (1960), this 


antibiotic (rhodomycetin), which 
active against gram-negative bac- 


strain shows much similarity, on the basis of 
phage sensitivity and other properties, to 
S. californicus. A detailed study of degenera- 


142 THE ACTINON 


IYCETES, Vol. II 


TABLE 21 


Classification of actinomycin-producing organisms based upon utilization of rhamnose and 


raffinose (Bt 


tlinger et al., 1956) 


Utilization of: 


Actinomycin Species Reference or origin of strain 
Rhamnose Raffinose 
+ S. flavovirens Pridham and Gottlieb + + 
+ S. flaveolus Kurosawa + + 
oo S. antibioticus Pridham and Gottlieb + = 
+ S. antibioticus Burkholder e¢ al. + = 
xX S. antibioticus orig. (Waksman) + = 
XE S. antibioticus NRRL (Raper) + — 
C S. chrysomallus orig. (Lindenbein) + = 
_ S. flaveolus orig. (Waksman) + = 
I S. parvullus orig. (Waksman) + — 
xX S. parvus NRRL (Benedict) + — 
x 3 ETH strains _ 
C 8 ETH strains a - 
x YTH 9001 _ _ 
TABLE 22 
Grouping of actinomycin-producing organisms (Ettlinger et al., 1956) 

Group arena Spore color ee Authentic strains ETH strains 

I + cinereus x S. antibioticus (orig. and 33 
NRRL) 
II + griseus 1 
. S. parv s (orig. al 
III is mcans ' parvullus (orig.) : 
( S. chrysomallus (orig. ) 17 
IV — griseus is) 
[ S. parvus (NRRL) 


tion and regeneration of S. griseus was made 
by Williams and McCoy (1953). 

Different varieties of S. griseus vary 
greatly in their cross-resistance and in their 
sensitivity to actinophages. 

It must further be noted that high-yielding 
streptomycin strains can be obtained by 
irradiation, by growth in media containing 
increasing concentrations of streptomycin, 
and by strain selection. 

Okami (1950a) examined 47 strains be- 
longing to the Griseus series. Five of these 
were grisein-forming strains and four were 


pink pigmented forms. The streptomycin- 
producing strains grew in maltose-containing 
media with NaNQOs: as a source of nitrogen, 
but not in glucose, glycerol, or sucrose media. 
The grisein strains grew in media containing 
any of the four carbon compounds. The pink 
strains grew only in glycerol media (Table 
23). The utilization of the carbohydrate was 
found to depend largely on the nitrogen 
source. In the presence of ammonium sulfate, 
the above differences disappeared. All strains 
utilized xylose, but not raffinose or rham- 
hose. Sensitivity to phage was said to be 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


strain specific, but not characteristic of 
streptomycin production. The use of strep- 
tomycin-resistant and — streptomycin-de- 
pendent cultures of bacteria as test organ- 
isms for the differentiation of the various 
strains of S. griseus was considered as supple- 
mentary to the foregoing differentiation 
methods. 

It is of particular interest, in this con- 
nection, to draw attention to the confusion 
that has arisen in some cases from Krassil- 
nikov’s attempt to change the name of the 
streptomycin-producing organism. Just as 
Waksman did previously, Krassilnikov came 
to the conclusion that there is a difference 
between the Krainsky and the Waksman 
and Curtis cultures of A. gréseus. Although 
he, likewise, had no opportunity to compare 
Krainsky’s original isolate with the strep- 
tomycin-producing organism, he attempted 
to draw conclusions on the basis of cultures 
that he isolated himself, and proposed that 
the name of the streptomycin-producing 
organism be changed to Actinomyces globi- 
sporus streptomycint (later changed to A. 
streptomycina). This suggested change was 
most unfortunate for several reasons: (a) a 
well described specific name, namely Strep- 
tomyces griseus Waksman and Henrici, was 
set aside merely for the sake of priority of a 
name of a culture (A. griseus Krainsky) 
which no one had ever seen and which was 
not available in any culture collection; (b) a 
hame of an organism that had become rec- 
ognized throughout the world because of its 
important physiological and biochemical 
properties, and especially because of its 
capacity to produce a highly important 
chemical substance, streptomycin, 
changed to a trinomial merely because of the 
existence of an insufficiently described vari- 
ety of an unknown culture. 

The confusion thus became compounded 
by this attempt to change the name of the 
streptomycin-producing organism. We find, 
in addition to the two names suggested by 


Was 


145 


TABLE 23 
Utilization of carbon sources by various strains of 
Streptomyces griseus with nitrate as source of 
nitrogen (Okami, 1950) 


Garbonsource Streptomycin Grisein Pink-pig- 
strain strain ment strain 
Glucose - + — 
Glycerol — + + 
Sucrose _ te — 
Maltose + + = 


KXrassiinikov, the incorrect names Actino- 
myces griseus Waksman listed by Koreniako 
and Nikitina (Shorin, 1957), Streptomyces 
griseus Krassilnikov by Znamenskaia et al. 
(1957), Actinomyces griseomycini by Gause 
et al. (1957), and finally Streptomyces glo- 
bisporus streptomycini by Severin and Gor- 
skaia (1957). This confusion was fortunately 
limited to the literature published in Rus- 
sian. 

For the reasons presented here, Krassil- 
nikov’s modifications of the name S. griseus, 
with all the subsequent confusing names, 
cannot be accepted. In fact, the actual 
culture, A. (Krainsky ) 
Krassilnikov (1949), belongs rather to the 
Cinereus series. 


Qriseus emend. 


The logical name for the streptomycin- 
producing 
griseus Waksman and Henrici. 


species remains Streptomyces 


Additional Organisms 


Numerous other organisms belonging to 
the Griseus series have been described as 
species and as varieties. Some of the descrip- 
tions are incomplete; and it is, therefore, 
rather difficult to give them an exact posi- 
tion. This is true, for example, of the cultures 
described by Gause et al. (1957) under the 
name A. 
other species placed in the series /Helvolus. 
See also Harada, 1959. 


rubiginosohelvolus and some of the 


IX. Series Hygroscopicus 


This series comprises organisms that form 
a white to gray aerial mycelium, with a 


144 


tendency to become dark gray; frequently 
black patches are produced in the mycelium, 
the whole often becoming black. The sub- 
strate growth is dark gray with a tendency to 
become moist, slimy, and finally changing 
to black. The species are melanin-negative, 
although on synthetic agar a brown to black 
soluble pigment may be produced. 

Morphologically the species give rise to 
spiral-shaped sporophores. This series com- 
prises a number of species, some of which 
are listed: S. endus, S. hygroscopicus, S. 
limosus, S. nigrificans, S. platensis, and S. 
violaceoniger. 


X. Series Scabies 


Characteristic Properties 


a. Sporophores produce spirals. 

b. Aerial mycelium lght gray to dark 
eray. 

c. Melanin-positive. 

d. Some species are able to cause diseases 
of plants, notably scab of potatoes. 

The melanin-producing capacity of certain 
actinomycetes, or their ability to form solu- 
ble brown to black pigments when grown in 
protein-containing media, was first recog- 
nized as a diagnostic characteristic by Rossi- 
Doria and Gasperini in 1891. Numerous 
cultures found capable of producing such 
pigments were isolated from different sub- 
strates, and designated as Streptothrix chro- 
mogena, Actinomyces chromogenus, or <A. 
chromogenes. Gradually it came to be recog- 
nized that all these isolates represented not 
a single species but a large number of organ- 
isms, differing greatly in their morphological, 
physiological, and biochemical properties. 

This was definitely established in 1900 by 
Beijerinck, who isolated two types of actino- 
mycetes (‘‘Streptothrix”’) represented abun- 
dantly in nature. “‘One of these that I have 
learned to recognize in the form of numerous 
varieties, I will designate as Str. chromogena 
Gasperini, since I believe that one such 


THE ACTINOMYCETES, Vol. II 


variety was available to the author of this 
name. The other species I designate as Str. 
alba.” The first was characterized by the 
formation of a brown pigment in meat ex- 
tract-gelatin media. 

Neukirch (1902) demonstrated the pres- 
ence in nature of two chromogenic types of 
actinomycetes. Krainsky (1914) described 
four chromogenic species, Whereas Waksman 
and Curtis (1916) demonstrated the occur- 
rence in soil of various other chromogenic 
types, “each with such well defined charac- 
ters as to make it almost impossible to 
classify them as one species.” 

One of the most important series among 
the chromogenic actinomycetes is S. scabies, 
which at one time was designated as S. 
chromogenus. In addition to the members of 
the Chromogenus series as such, numerous 
other species now included in other groups 
also possess chromogenic properties, espe- 
cially members of the Lavendulae and Re- 
ticult series. 

The soluble pigment produced by various 
organisms when grown on protein-containing 
media was found to belong to the melanin 
type. It frequently involved the tyrosinase 
reaction. The intensity of the pigment varies 
with the organism and with the medium. 
The formation of the melanin pigment is 
usually determined by growing the organ- 
isms on tyrosine-containing media. 

Baldacci et al. (1953) did not recognize a 
“Chromogenus” series as such, although they 
Gause et al. 


listed one under ‘‘Cas-Gri.” 
(1957) listed two series, one a “Chromogenus”’ 
proper, and the other ‘‘Helvolus,” in which 
both pigment-producing and nonpigment- 
producing types are included. 

The organisms belonging to S. scabies were 
at first believed to be primarily associated 
with scabbiness in white potatoes, sugar 
beets, and mangels. Only the typical chro- 
mogens were at first included in this series. 
They formed a brown to dark brown to black 
growth, a gray aerial mycelium, and a brown 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


to black soluble pigment on organic media. 
It must be conceded at once that not all 
organisms isolated from scabby potatoes or 
beets are able to produce a soluble brown 
pigment and certainly not all are capable of 
causing scabbiness in potatoes. 

Millard and Burr (1926) isolated a num- 
ber of cultures from scabby potatoes and 
beets. They proposed a key for the identifica- 
tion of the presumably potato-disease-pro- 
ducing actinomycetes (see Volume I, Chap- 
ter 18). The medium this 
purpose (glycerol nitrate solution) could 
hardly be considered the most desirable sub- 
strate for bringing out the proper characters 
for a system of classification. Some of these 
cultures, notably S. clavifer and S. fimbri- 
atus, definitely belong to the Scabies series. 

Baldacci and Spalla (1956) suggested that 
the strain of S. scabies isolated by Millard 
and Burr be designated as S. scabies var. 
anglica. It is distinguished from the North 
American species first described by Thaxter 
as having a “gray substrate growth, a gray 
aerial mycelium and a yellow soluble pig- 
ment.” 

The possibility that different strains or 
races of SS. scabies were responsible for the 
infection of potatoes and mangels has been 
fully recognized. No definite correlation has 
been found, however, between pathogenicity 
and cultural and other properties of the or- 
ganism, although variants may differ from 
the parent culture in pathogenicity. High 
nitrogen content of the medium appeared to 
inhibit production of aerial mycelium in the 
parasitic strains but not in the saprophytes. 
Of the 20 isolates tested by Schaal (1944) on 
three different media, six did not produce 
any spirals but 14 did. These spirals were of 


selected for 


both sinistrorse and dextrorse types. 

Taylor and Decker (1947), in a study of 
143 isolates obtained from scabby potatoes, 
beets, and radishes, the 
criteria for their classification: acid-fastness; 
starch hydrolysis; formation of dark brown 


used following 


145 


surface ring on milk; acidification of milk; 
reduction of nitrate to nitrite; utilization of 
certain sugars, organic acids, and paraffin: 
gelatin liquefaction; pigment formation 
from tyrosine; and maximum growth tem- 
perature. The only true correlation between 
specific cultural properties and the ability to 
produce typical lesions of potato scab was 
obtained in the production of a dark brown 
ring of surface growth on milk. 

The following species may be tentatively 
included in the Scabies series: S. scabies, S. 
hawaviensis, and S. galtieri. 

A number of other organisms isolated from 
potato tubers or directly from the soil were 
found capable of causing scab of potatoes 
and must be included in this series. This is 
true, for example, of A. violaceochromogenes 
described by Krassilnikov (1949), and of A. 
chromofuscus and A. prunicolor of Gause et 
al. (1957). Other closely related forms have 
been described, although pathogenicity tests 
were not made. 

A number of forms that apparently have 
nothing to do with scab formation, but have 
the characteristic properties of the series may 
also be included. 


XL. Series Lavendulae 


Characteristic Properties 

a. Sporophores straight or spiral-forming; 
spores oval, smooth surface. 

b. Aerial mycelium colored lavender to 
pale blue. 

c. Melanin-positive. 

This is one of the true chromogenic series 
of the genus Streptomyces. Organisms be- 
longing to the Lavendulae series are widely 
distributed in the soil and are represented 
there by a large number of species and varie- 
ties. Many of them are strongly antagonistic 
and are capable of forming various important 
antibiotics, such as streptothricin. Wood- 
ruff and McDaniel (1958) reported that 90 
per cent of all the antibiotics produced by 


146 


actinomycetes are streptothricins; here be- 
long various related compounds, such as 
streptin, streptolin, actinorubin, and anti- 
biotic 136. 

The most important species belonging to 
this series are S. lavendulae and S. venezuelae, 
organisms producing streptothricin and 
chloramphenicol respectively. 

S. lavendulae comprises organisms ex- 
tremely variable in nature. Many of them 
give rise, on cultivation, to different variants 
or mutants. Some of these variants produce 
a blue diffusible pigment on glucose-peptone 
agar; others form a brown pigment. The sub- 
strate mycelium of the blue pigment-forming 
variants is pale blue, with scattered, small 
pinpoint areas of deep blue. Upon complete 
sporulation, the substrate growth becomes 
covered with the characteristic lavender- 
colored aerial mycelium; occasional sunken 
areas have a slightly bluish tinge; the reverse 
of the substrate growth is cream-colored 
except for the small blue spots. Other vari- 
ants produce a colorless to cream-colored 
substrate growth free of any blue pigment 
whatsoever; a brown diffusible pigment ap- 
pears later, and the growth becomes covered 
with thick lavender-colored mycelium. The 
two types of variants are stable in nature. 
Some variants may lose the capacity to 
produce aerial mycelium. 

S. venezuelae, as well, gives rise to a num- 
ber of variants. Two strains were isolated 
and found to be similar to S. lavendulae in 
their cultural and physiological properties, 
although they differed in their ability to 
utilize various carbohydrates. Streptomyces 
venezuelae utilizes arabinose, rhamnose, 
xylose, lactose, and fructose; S. lavendulae 
has either no effect or only a limited effect 
upon these carbohydrates. The former also 
differs from the latter in its sensitivity to 
actinophage and in various serological re- 
actions. 

Streptomyces venezuelae was described as 
having a thin-walled substrate mycelium, 
colorless, hyaline, monopodially branched, 


THE ACTINOMYCETES, Vol. II 


the hyphae varying in diameter from 0.9 to 
1.8 wand the branches growing to about 150 
uw in length. The aerial mycelium appears 
lavender under the microscope, thick-walled, 
generally not much branched, straight or 
shghtly and irregularly curved, not forming 
spirals, individual hyphae arising frequently 
from the primary mycelium at the surface 
of the substrate. The color of the colonies, 
when viewed on agar without magnification, 
is gray to light tan or pink, but not lavender. 
The upper portions of the aerial hyphae 
divide into chains of spores. These are oval 
to oblong 0.4 to 0.9 by 0.7 to 1.6 wu. Individ- 
ual spores are colorless at maturity, but in 
mass appear tan to gray when viewed with- 
out magnification. 

Okami (1956) made a comparative study 
of the organisms commonly included in the 
Lavendulae series on the basis of the color of 
the aerial mycelum and certain other char- 
acteristics. He found that eight cultures, 
notably the streptothricin-producing forms, 
possessed the following properties which he 
considered as standard for the series. 

a. Aerial mycelium pink-lavender color 
when grown on yeast extract-glucose 
agar. 

b. Brown pigment when grown on yeast 
extract-glucose agar. 

c. Very sensitive to chlortetracycline and 
chloramphenicol; relatively sensitive to 
streptomycin; relatively resistant to 
neomycin; and resistant to strepto- 
thricin. 

d. Utilize: glucose, 
mannose. Do not utilize: arabinose, 
fructose, lactose, mannitol, raffinose, 


galactose, maltose, 


rhamnose, sucrose, xylose. 
e. They show certain growth-inhibiting 
effects (Tables 24 and 25). 

On continued cultivation for 40 years on 
artificial media, the original 1915 isolate of 
S. lavendulae (No. 3330) lost many of its 
characteristic properties: 

1. It no longer produced any aerial my- 

celium. 


SERIES AND SPECIES OF 


2. It did not form any dark brown pig- 
ment. 
3. It was now able to utilize fructose, 
mannitol, rhamnose, xylose. 
4. It showed no or very weak antagonistic 
action. 
On the basis of the above properties, 
Okami divided the S. lavendulae series into 
10 subgroups (Table 26). 


TABLE 24 
Antibacterial activity of different strains of 


Streptomyces lavendulae (Okami, 1956) 


Sl lavendulae Inhibition zone, test bacteria 


strain no. = E 


B. cereus 


y. coli Staph. aureus 

mm mm mm 
3330 0 0 0 
3440-8 1t7/ 16 2 
3440-14 6 6 2 
3516 1S lg 2 
3516-W 10 10 2 
3530 16 16 2 
3531 0 0 0 
3532 27 23 18 
3542 18 17 fel 
3543 11 1s 6 
3508 0) 0 i) 
3445 0 0 0 
3465 2 4 5 
3544 11 i) 3 
3559 21 


GENUS STREPTOMYCES 


considered S. lavendulae as a 


Krassilnikov, 


Baldacci 
distinct group. 
looked upon these organisms as members of 
the ‘‘chromogenus” group. et al. 
created a series under the name ‘‘/avendulae- 
and subdivided it 
groups, based upon the formation of a solu- 
ble pigment in organic media: (1) The first 
included cultures that formed 
pigment, such as S. virginiae; (2) The second 
included SS. venezuelae, S. lavendulae, and S. 
circulatus, as well as a variety of others, all 
of which formed a brown to black pigment 
in organic media; (3) The third produced a 
vellow pigment. As the requirements for the 
group S. lavendulae presented above indicate, 
neither the first nor the third of these sub- 
groups belong to this group. 

Sanchez-Marroquin (1958) found that 
three related species of Streptomyces produc- 
ing a pink to lavender aerial mycelium on 
synthetic media could be distinguished from 
one another as follows: (a) S. fradzae pro- 


however, 
(Gaause 


roseus”’ into three sub- 


no soluble 


duces spirals on short branches; (b) S. 
lavendulae forms spirals at the tip of long 
straight branches; (c) S. venezuelae forms no 
spirals, but only straight sporophores; the 
first is nonchromogenic and the last two 
are chromogenic. The Lavendulae series was 


TABLE 25 


Effect of composition of medium on reciprocal antagonism between different strains of 


Streptomyces lavendulae (Okami, 1956) 


Inhibition zone of S. lavendulae 3516 


Inhibition zone of S. lavendulae 3440-8 


Strain number Agar media* 


Agar media 


YGA == = - Soya YDA ———__—_—___—— Soya 
ASP Starch ASP Starch 

mm mm mm mm mm mm mm mm 
3330 4.0 5.0 7.0 4.0 3.0 0 2.0 3.0 
3440-8 17.0 15.0 20.0 15.0 0) i) 0) 0 
3440-14 17.0 4.0 18.0 10.0 0 0 0 0 
3516 0 0.5 0 0 0 0 0 0 
3516-W 0 2.0 3.0 1.0 0 0 0 0 
3530 0 1.0 1.0 1.0 0 0 0 0 
3531 12.0 1.0 5.0 8.0 0 0 0 0 
3532 16.0 10.0 2.0 12.0 7.0 0 10.0 10.0 

* YGA = yeast extract-glucose; ASP = asparagine-glucose. 


148 


THE ACTINOMYCETES, Vol. II 


TABLE 26 


Classification of the lavendulae series into subgroups (Okami, 1956) 
Subgroup Culture No. Characteristics 
A Standard S. lavendulae 
3440-8 a. Aerial mycelium with pink-lavender color on YDA 
3440-14 b. Brown pigment on YDA 
3516 c. Very sensitive to chlortetracycline and chloramphenicol, relatively 
3516-W sensitive to streptomycin, relatively resistant to neomycin, resistant to 
3530 streptothricin 
3542 d. Utilizes: galactose, maltose, mannose 
3544 Does not utilize arabinose, fructose, lactose, mannitol, raffinose, rham- 
3555 nose, sucrose, xylose 
e. Shows certain inhibiting effects 
B 3330 a. No aerial mycelium 
b. No dark brown pigment 
d. Utilizes fructose, rhamnose, mannitol, xylose 
e. Antagonistic action none or very weak 
C 3531 c. Relatively resistant to chloramphenicol 
D 3532 c. Relatively resistant to chlortetracycline 
e. Antagonistic action none or weak 
E 3543 b. No dark brown pigment 
c. Relatively resistant to chloramphenicol 
d. Utilizes arabinose, fructose, lactose, mannitol, raffinose, rhamnose, su- 
crose, xylose 
e. Antagonistic action none or weak 
F 3568 d. Utilizes arabinose 
e. Antagonistic action none or weak 
G 3465 b. No dark brown pigment 
e. Antagonistic action none or weak 
H 3555 c. Relatively sensitive to streptothricin VI 
d. Utilizes fructose 
e. Antagonistic action none or weak 
I 3625 c. Resistant to chloramphenicol 
3534 d. Utilizes arabinose, rhamnose, xylose 
3534-A e. Antagonistic action none or weak 
J 3651* d. Does not utilize mannose 
3652* 


* S. virginiae 


said to include not only S. lavendulae but also 
S. cinnamonensis and SS. virginiae. 

To indicate the confusion in classifying 
of the series, It 1s 


members Lavendulae 


suficient to cite the work of Kuchayeva 
(1958). She collected 22 cultures, freshly 
isolated or obtained from different labora- 
tories and believed to belong to this group. 


— 


SERIES AND SPECIES OF GENUS STREPTOMYCES 


Fight of the cultures produced straight 
sporophores and 14 formed  spiral-shaped 
sporophores. The color of the aerial myce- 
lium of the substrate growth varied from 
vellow to reddish-brown. Some produced a 
melanin pigment and others did not, thus 
automatically excluding the last as members 
of the Lavendulae series. They varied greatly 
in their antagonistic properties, some in- 
hibiting the growth of all bacteria and fungi 
tested, and others having no effect either on 
gram-negative bacteria or on certain fungi. 
The following antibiotics were listed as 
products of the Lavendulae series, thus sug- 
gesting the possible specific differences in 
the series: streptothricin, streptin, antismeg- 
matis factor, antibiotic 136, lavendulin, 
actinorubin, pleocidin, ehrlichin, actithiazic 
acid, antibiotic MD 2428, and grasseriomy- 
cin. 


XIL. Series Erythrochromogenes 


This is a large melanin-positive series of 
organisms. The aerial mycelium is usually 
white with a yellowish or brownish shade to 
gray with a bluish or greenish shade; it is 
often reddish to The 
growth is colorless to orange to red or even 
brown to black. Certain species placed in 
this group often produce a greenish-yellow 
or reddish-brown to almost black soluble 


brown. substrate 


pigment on synthetic media. The sporo- 
phores are straight or produce spirals. 

This melanin-positive series comprises a 
large number of species, such as represented 
by the following: S. erythrochromogenes, S. 
bobiliae, and S. cinereoruber. 


XIIL. Series Viridochromogenes 


This melanin-positive series of organisms 
is characterized by an ash-gray to greenish 
to olive-green to bluish aerial mycelium. 
The substrate growth is cream-colored to 
brown to greenish to black. Soluble pigment 
on inorganic media is yellowish to greenish 


149 


to black. On organic media, soluble pigment 
is brown to deep brown to olive or purple or 
black. 

The sporophores form spirals. 

This series is widely represented in nature 
by a number of species, namely: S. virido- 
chromogenes, S. chartreusis, and S. cyaneus. 


XIV. Series Cinnamomeus 


Characteristic Properties 
a. Sporophores produced in verticils. 
b. Aerial mycelium white, 
pinkish. 


vellow, or 


c. Substrate growth yellowish to pinkish. 

d. Melanin-negative. 

This series is characterized by the forma- 
tion of verticil-bearing sporophores. The 
verticils are both primary and secondary. 
Spirals usually are not produced, although 
occasionally some spirals are formed. The 
species within this series are melanin-nega- 
tive, although a purplish pigment may be 
produced on certain organic media. The 
aerial mycelium is white to pinkish to cin- 
namon-colored. The substrate growth is 
vellowish to brown to pinkish. 

The species included in this series can be 
listed here: S. hachijoensis, S. fervens, and 
S. cinnamomeus. 


XV. Series Reticuli 


Characteristic Properties 
I 


a. Sporophores produced in verticils, 
straight or spiral-shaped, on the primary or 
secondary aerial hyphae. 

b. Aerial mycelium white to gray. 

c. Melanin-positive. 

This is one of the two series within the 
genus Streptomyces which are differentiated 
from the other members of the genus pri- 
marily by their morphology. Species within 
this series are characterized by the radial 
arrangement of the sporophores, whereby 


three or more branches originate from a 


150 


node, thus forming a ‘‘verticil,”’ frequently 
referred to as a ‘‘whorl.’? Very often the 
sporophores may be branched toward the 
end of the sporulating-hyphae, giving the 
appearance of a “broom shaped branch,”’ 
or may give rise to the formation of a 
“cluster,” which is to be distinguished from 
a typical verticil of sporophores. The verti- 
cils may be primary or secondary in nature. 
Among the variations of a verticil is the 
formation of tufts, when straight branches 
are grouped together on the aerial hyphae. 
The formation and nature of the verticils 
may be changed with the composition of the 
medium, a phenomenon first reported by 
Waksman and Curtis (1916) and more 
recently by Nakazawa (1955) and Shinobu 
(1955a). Both primary and secondary ver- 
ticils may be formed in the same culture. 
Baldacci (1953) did not recognize this im- 
portant series at all. He gave series recog- 
nition to one of its members, S. rubrireticult, 
merely on the basis of its pigmentation. 
Gause et al. (1957), following this example, 
included in the ‘ruber’ series one ‘‘tuft- 
forming” organism, also on the basis of its 
pigmentation. Later, however, Baldacci 
(1958) suggested separation of the verticil- 
producing organisms into a separate genus, 
Streptoverticillium, as 
(Chapter 4, Volume I). 
Various investigators have used morphol- 


shown — elsewhere 


ogy as a basis for series separation of the 

genus Streptomyces. It is sufficient to list 

here three of them. 

Shinobu (1955a) proposed division of the 

genus as follows: 

1. Monopodial branching. This section has 
been divided on the basis of spiral forma- 
tion. 

2. Verticil formation: 

a. Nitella type. Typical radial branches 
almost equal in length. No spirals 
formed. S. reticulz is given as a typical 
representative. 


b. Anitella type. Radial branches differ 


THE ACTINOMYCETES, Vol. II 


from each other. Spirals formed with- 
out proper radial symmetry. S. virido- 
chromogenes is representative. 
The S. reticuli subgroup was further di- 
vided into: 


A. Verticils only primary. Sporophores straight. 
Streptomyces verticillatus 
Streptomyces hiroshimensis 
B. Verticils both primary and secondary. Sporo- 
phores form spirals. 
I. Colorless to brownish growth on synthetie 
media. 
1. Good growth on protein media. Spores 
spherical, oval. 
Streptomyces reticult 
Streptomyces albireticuli 
2. Poor growth on protein media. Spores 
cylindrical. 
Streptomyces circulatus 
II. Growth on synthetic media pink to red. 
Streptomyces reticuloruber 
Streptomyces griseocarneus 
Ill. Growth on synthetic media greenish. 
Streptomyces verticilloviridans 


Solovieva et al. (1957) made a study of 
cultures belonging to the S. reticuli series 
and isolated from Pamir soils. These cultures 
were divided into two subgroups: 

I. S. verticillatus, with straight sporophores 
(primary and secondary). 
Il. S. reticulz, with spiral-shaped  sporo- 
phores. 

These subgroups showed very little dif- 
ference in their physiological and biochem- 
ical properties. Only one strain, S. rubri- 
reticuli, showed some differences (weaker 
gelatin liquefaction, strong nitrate reduction, 
weak growth on cellulose). From an anti- 
biotic point of view, however, there was a 
marked difference; members of the first sub- 
group showed strong antifungal activity, 
whereas subgroup II gave weaker activity 
or none. 

Pridham et al. (1958) divided the verticil- 
forming series into four morphological sub- 


groups: 
1. Monoverticillate, no spirals. 
2. Monoverticillate, with spirals. 


SERIES AND SPECIES OF 


3. Biverticillate, no spirals. 
4. Biverticillate, with spirals. 

The following species were included in this 
series: S. reticuli, S. netropsis, S. thioluteus, 
S. griseocarneus, and S. verticillatus. 

Unfortunately, some cultures show both 
straight and — spiral-shaped 
Nomi (1960) criticized the above system; 
he could not accept the spiral-producing 
forms among the true verticil types. He con- 
sidered the group to consist of the typical 


sporophores. 


“biverticils’”” comprising both primary and 
secondary elements; the atypical ‘‘mono- 
verticils’ comprising primary — verticils, 
mixed verticils, and sometimes more com- 
pound polyverticils. He included the follow- 
ing species: S. reticult, S. hiroshimensis, S. 
albireticuli, S. echimensis, S. griseocarneus, 
S. thioluteus, S. salmonicida, and 8. netropsis. 


GENUS STREPTOMYCES 15] 


Series Thermophilus is discussed in Chap- 
ter 11, among the thermophilic  actino- 


mycetes. 


Other Possible Series 


In addition to these 16 series of the genus 
Streptomyces, other series could be suggested, 
based either on the color of the aerial myce- 
lium or of the substrate growth; ecological 
or physiological criteria have also been pro- 
posed for series characterization. Certain 
groups, such as the thermophilic forms be- 
longing to the genus Streptomyces are in- 
cluded in a special series (Vhermophilus), 
and are discussed elsewhere (Chapter 10), 
since they are definitely related, because of 
their close ecological and physiological re- 
lationships, to the other thermophilic genera. 


Cohva'p teu, 77 


Classification of Streptomyces Species 


The difficulties encountered by an inex- 
perienced investigator in identifying a 
freshly isolated culture of a Streptomyces with 
previously described species have been 
brought out in the preceding three chapters. 
No wonder an inexperienced worker soon 
becomes discouraged and takes the easy path 
of creating a new species or variety for 
every newly isolated culture. This is es- 
pecially true if such a culture produces an 
antibiotic not previously described or an 
apparently different form of a known anti- 
biotic that he is anxious to patent or on 
which he wishes to establish priority. Among 
the significant factors that have contributed 
to this rash of “new” species are: 

1. Inadequate description of previously 
described species with which comparisons 
are made. 

2. Overlapping of the morphological and 
cultural characteristics of strains or species 
previously described. 

3. Variations in composition of the media 
used in describing species. 

4. Failure to recognize natural variability 
of different strains that might be meluded 
in a single species. 

5. Idiosynerasies of the particular inves- 
tigator, and his tendency to be either a 
“Jumper” or a “‘splitter.”’ 

In spite of these discouraging aspects of 
the problem of identification of particular 
organisms, Classification and characteriza- 
tion of Streptomyces species have recently 
made considerable progress. Though various 
criticisms have been directed toward it, the 


system of classification of actinomycetes 
used in the seven editions of Bergey’s Man- 
ual still appears to be the most logical and 
most workable, except for certain modifica- 
tions that are now desirable. In every new 
edition, advantage has been taken of the 
accumulated information to modify this sys- 
tem of classification, especially of the genus 
Streptomyces. In this treatise, a complete re- 
arrangement has been made in classifying 
the species included in the genus Strepto- 
myces, as compared with the last (seventh) 
edition of Bergey’s Manual. 

The thermophilic species of Streptomyces 
have been placed in a separate series Thermo- 


philus and transferred to Chapter 11, in 
which all the thermophilic actinomycetes 


are included. Species of Streptomyces 1so- 
lated from animal and plant infections, es- 
pecially those for which pathogenicity has 
not been established, have been distributed 
throughout the genus, thus removing the 
need for a major separation of these species 
into saprophytic versus parasitic forms. 
These properties are now given only sec- 
ondary consideration in characterizing spe- 
cies within the genus. It has further been 
recognized and emphasized, time and again, 
that it is most desirable to utilize morpho- 
logical properties in defining and charac- 
terizing species of Streptomyces, though in 
some instances this could not be done with 
any degree of assurance. 

New genera have been created, once cer- 
tain well-defined morphological and physio- 
logical properties suggested its advisability. 


: 


CLASSIFICATION OF STREPTOMYCES SPECIES 


Whether this practice should be extended 
and recognition thus be given to the capacity 
to form sclerotia or to the ability to form 
verticils by certain Streptomyces species, 
thus placing them in separate genera, re- 
mains to be determined. The author’s sug- 
gestion of many years ago that the structure 
of the sporophores (straight versus spiral- 
forming, closed versus open spirals, tuft- and 
verticil-producing) be used in characterizing 
certain species or species-groups is gaining 
wider recognition, though some investiga- 
tors do not consider this a sufficiently con- 
stant property for the major subdivision of 
the genus and suggest that it be left for 
secondary characterization. The shape and 
size of the spores appear to be less signifi- 
‘ant properties, although the surface of the 
spores, as detected by the electron micro- 
scope, has been gaining approval. 

Among the most annoying characteristics 
of the genus Streptomyces are: (a) the loss of 
capacity by certain species to produce aerial 
mycelium, and (b) the overlapping property 
on the part of certain species of Nocardia to 
produce an aerial mycelium that cannot be 
differentiated from that of Streptomyces. It is 
true, however, that cultures of Streptomyces, 
even if they have lost the capacity to pro- 
duce aerial mycelium, can still be recognized 
by the structure of their substrate mycelium 
and by certain cultural and physiological 
properties. The latter include the nature of 


their soluble pigments, their ability to 
liquefy gelatin, hydrolyze starch, invert 


sucrose, and coagulate and peptonize milk. 
Some strains that have lost the capacity to 
produce aerial mycelium may regain this 
property if they are grown in sterile soil or 
in special soil media, or are subjected to 
other special treatments. 

The growth of cultures of Streptomyces 
that have lost the capacity to produce aerial 
mycelium is often colorless, though some- 
times pigmented; it is smooth or lichenoid, 
leathery, compact, with a shiny surface. 


Some produce a soluble brown pigment. 
Some are able to form antibiotics. On the 
assumption that such cultures, because they 
do not form aerial spores, should be con- 
sidered as sterile, Krassilnikov designates 
them as trmomials with a third component 
of the name “‘sterilis.’”’ This is analogous to 
Fungus sterilis among the fungi. Certain 
such species are included in the present 
classification; others may be considered as 
typical nocardias and have been transferred 
to that genus. 


Characterization of Streptomyces Spe- 
cies 


Among the properties to receive major 
consideration in describing individual spe- 
cies are the following: 

1. Morphological properties: These include 
formation and nature of substrate (vegeta- 
tive) growth and of aerial mycelium, manner 
of sporulation (spiral formation, verticil 
formation), nature and surface of spores. 

2. Cultural characteristics. These 
prise color and color changes of substrate 
growth and of aerial mycelium, and forma- 
tion of soluble pigments on synthetie and 


com- 


organic media. The most significant of these 
pigments are the melanins or melanoids pro- 
duced in media containing tyrosine or pro- 
teins and peptones. The brown to black 
pigments produced in such media by certain 
species of Streptomyces are said to designate 
melanin-positive as opposed to melanin- 
negative reactions, or tyrosinase-positive 
versus tyrosinase-negative reactions. In view 
of the fact, however, that few of the older 
investigators tested this reaction in tyrosine— 
free media, it is more desirable to use the 
designations chromogenic (melanin +) or 
nonchromogenic (melanin —). In this case, 
chromogenicity the 
formation of brown to black pigments on 


refers specifically to 


protein-containing media. 
3. Physiological and biochemical properties. 
These include: proteolytic activities, such as 


154 


gelatin liquefaction, coagulation and pep- 
tonization of milk, and hemolysis of blood; 
utilization of carbon compounds; antagonis- 
tic properties and formation of antibiotics; 
effect of temperature, aeration, and reaction 
upon growth; formation of specific enzymes, 
such as oxidase, lipase, invertase, diastase, 
mannase and protease; reduction of nitrate, 
and formation of H.S. There is no sharp line 
of demarcation between cultural and physio- 
logical properties, on the one hand, and be- 
tween physiological and biochemical activi- 
ties, on the other. 

4. Ecology. The ability of the organism to 
cause animal or plant diseases, and its occur- 
rence in a natural environment are important 
characteristics. 

5. Supplementary characteristics. Addi- 
tional characteristics that may be utilized 
for descriptive purposes include: (a) sero- 
logical reactions, (b) phage sensitivity, and 
(c) sensitivity to specific antibiotics. 

In describing the various species, one has 
to depend frequently upon the mformation 
supplied in published reports, since type 
cultures often are not available. Where the 
desired information is lacking or where the 
description, for various reasons, Is iInade- 
quate, the species may be placed in the list 
of incompletely described forms. 

tecently there has been a tendency to 
overlook earlier described species, and to 
emphasize and often give new names to 
newly isolated cultures. One cannot con- 
demn this tendency too strongly. While, m 
most cases, it is dificult to establish synon- 
vmy because of a lack of type cultures or a 
possible change in such cultures upon pro- 
longed cultivation on artificial media, every 
effort must be made to give credit to the 
sarlier investigator. One has no patience, 
therefore, with those attempts to set aside, 
willfully or unwillfully, older descriptions 


or to consider such organisms as varieties of 


newly isolated and newly named cultures. 
When a culture is freshly isolated, a study 


THE ACTINOMYCETES, Vol. II 


should be made both of its position in a 
particular group in the genus and of its 
classification as a species. Only by a com- 
bination of properties described under both 
can one determine the identity of the new 
culture. Obviously, no conclusions should be 
drawn that such a culture represents a new 
species merely on the basis of certain super- 
ficial observations, such as a delay in coag- 
ulation and peptonization of milk or in 
liquefaction of gelatin, or because of a differ- 
ence in the intensity of coloration of the 
potato plug, or in the shade of pigmentation 
of growth on a particular medium, or even 
in the degree of curvature of the sporophores. 
Any attempt to create new species on the 
basis of such minor variations must be con- 
sidered both unscientific and confusing. The 
tests must be repeated again and again to 
confirm the recorded observations. 

The need for a knowledge of the exact 
composition of the media used for descrip- 
tive purposes can hardly be overemphasized. 
Some species show their most characteristic 
property upon only one particular medium, 
and unless such a medium is used, the prop- 
erties of a new isolate can easily be over- 
looked. S. fradiae, for example, shows the 
characteristic color (seashell-pink) of its 
aerial mycelium upon potato-starch agar. 
Certain synthetic agar media, notably 
nitrate-sucrose, glucose-asparagine, and cal- 
cium malate, are among those which alone 
bring out characteristic properties of certain 
other species. Growth on potato is quite 
characteristic, although the variety of the 
potato, the manner of crop fertilization, and 
other factors may influence the nature of the 
growth of the organism and pigment formed. 

In view of the great interest at the present 
time in the screening programs for antibi- 
oties, when literally many thousands of cul- 
tures are being isolated and tested, there is 
naturally a tendency on the part of some 
investigators to consider the ability of a 
particular culture to form a specific anti- 


CLASSIFICATION OF STREPTOMYCES SPECIES 


biotic as its major characteristic property. 
But this capacity is often a strain rather than 
a species characteristic. In view of the mu- 
tational possibilities of such cultures and of 
the marked effect of composition of medium 
and environmental factors upon the qualita- 
tive nature and quantitative yield of the 
antibiotic, one must consider such a prop- 
erty, at best, as only a secondary character- 
istic and avoid assigning to it an important 
role in creating new species. 

Shinobu (1958b) emphasized again that 
only synthetic media should be used for the 
study of the sporulation of the aerial myce- 
lium, notably spiral formation, in Strepto- 
myces. In most species, the curvature of the 
spiral is sinistrorse (counterclockwise); In a 
few, dextrorse (clockwise). The diameter of 
the spiral varies from 1.5 to 8.0 uw and is not 
a characteristic property, although it is fixed 
for some species. As pointed out previously 
(Chapter 4), three morphological groups 
were recognized: (1) Those forming straight 
or wavy aerial mycelium, (2) Spiral-forming 
types, and (3) Verticil-forming types. 

Other criteria have been suggested. Some 
of these may be utilized for supplementary 
information in describing species and vari- 
eties. A Subcommittee on Actinomycetes of 
the Society of American Bacteriologists 
(Gottlieb, 1960) gave careful consideration 
to the various criteria used in describing and 
characterizing species of Streptomyces. They 
‘ame to the conclusion that morphology is 
to be considered as one of the more impor- 
tant criteria. Color of the aerial mycelium 
and carbon utilization are also important. 
Supplementary characteristics are provided 
by the production of HS, reduction of 
nitrate, and gelatin liquefaction. Such cri- 
teria as color of substrate growth and nitro- 
gen utilization were not considered of suffi- 
cient significance in describing new species. 

Many species, including a number of 
newly isolated forms, have been placed in 
Chapter 13 as incompletely described. Others 


— 
On 
Or 


have been listed as synonyms. Care has been 
taken to avoid the creation of many 


varieties, unless it has been fully established 


new 


that such varieties have a sound morpho- 
logical or cultural Whether 
varieties should be raised to the status of 
species remains to be determined by further 
study. Any such attempt would automat- 
ically lead to the temptation to create new 
species out of mutants, which are unfortu- 
nately altogether whether 
naturally occurring or artificially created. 
For the time being, they may still be con- 
sidered as varieties. 


basis. these 


too common, 


Classification of Genus Streptomyces 


In proposing the present system of clas- 
sification of the genus Streptomyces, the 
following properties have been given the 
greatest consideration: 

lL. Morphology of sporulating bodies; size, 
shape, and surface of spores. 

2. Color of aerial mycelium and of sub- 
strate growth. 

3. Color of growth. 

4. Formation of soluble chromogenic or 
melanoid pigments in proteinaceous media. 
This property is used, together with micro- 
morphology, for the major subdivisions of 
the genus. 

5. Formation of soluble pigments in syn- 
thetic media. 

6. Certain biochemical properties, notably 
proteolysis, starch hydrolysis, nitrate reduc- 
tion, formation of HS, utilization of carbon 
sources, and formation of specific anti- 
biotics. 

This system is a modification of the one 
originally used by Waksman and Curtis in 
1916, variously changed in subsequent years 
by Waksman and by Jensen, and used in 
the various editions of 
Bac- 


modified forms in 
Bergey’s Manual of Determinative 
teriology. 

A. Sporophores straight, wavy, or spiral-shaped; 


no verticils. 


156 THE ACTINOMYCETES, 


I. Proteinaceous mediaare not pigmented deep 
brown or black; melanin—negative. Soluble 
pigment on various media is faint brown, 
pink, red, purple, yellow, blue, or absent. 

1. Soluble pigment only faint yellow or 
faint brown. 
a. Aerial mycelium white, abundant. 
al. Sporophores produce spirals. 
a®. Occurs in soil and in certain 
other natural substrates. 
15. Streptomyces albus 
b?. Occurs in the sea. 
148. Streptomyces marinus 
b!. Sporophores straight. 
a2, Sporophores produce broom- 
shaped clusters. 
102. Streptomyces globispo- 
rus 
b2. No clusters produced. 
a*. Nonproteolytic. 
27. Streptomyces  autotro- 
phicus 
b’. Weakly proteolytic. 
179. Streptomyces orientalis 
ec’. Strongly proteolytic. 
129. Streptomyces kimbert 

b. Aerial mycelium, white, scant. 

250. Streptomyces willmoret 

c. Aerial mycelium white to light gray. 
al. Sporophores produce compact 

spirals. 
a®. Growth colorless. 
18. Streptomyces annula- 
tus 


— 
~ 
[<) 


2, Growth colorless to yellow- 
ish. 
92. Streptomyces  fungict- 


dicus 


io) 
t 


Growth brown to. orange- 
brown. 
21. Streptomyces arenae 
d?. No growth on sucrose nitrate 
agar. 
22. Streptomyces argenteo- 
lus 
b!. Sporophores form loose spirals. 
40. Streptomyces calvus 
c!. Sporophores straight or wavy. 
a®. Growth on sucrose nitrate 
agar yellowish-brown. 
2. Streptomyces — abura- 
vieNnsts 
b?. Growth gray to greenish. 
60. Streptomyces — coroni- 
formis 


Vol-vil 


e2. Growth vellowish-white; 
utilizes paraffin and rubber. 
68. Streptomyces elasticus 


. Aerial mycelium white to mouse- 


gray; spores bluish-gray. 
al. Sporophores produce compact 
spirals. 
227. Streptomyces spheroi- 
des 
b!. Sporophores in clusters; a few 
compact spirals. 
45. Streptomyces catenulae 
c!. Sporophores produce open cork- 
screw spirals. 
a®. Soluble pigment yellow. 
a’. Growth on — synthetic 
media cream-colored. 
167. Streptomyces niveus 
b*. Growth on synthetic me- 
dia vellow. 
143. Streptomyces — macro- 
sporeus 
b?. Soluble pigment tan to 
brown. 
35. Streptomyces caelestis 
!| Sporophores straight. 
111. Streptomyces griseolus 
Aerial mycelium white to gray, 
covered with dark humid stains or 
guttation drops. 
a'. Sporophores form spirals. 
a®. Growth buff to olive-colored. 
189. Streptomyces platensis 
b?. Growth colorless. 
123. Streptomyces humidus 
b!. Sporophores straight. 
a®. Growth on potato cream- 
colored. 


+ 
pou 


61. Streptomyces craterifer 
b?. Growth on potato slimy to 
black. 
235. Streptomyces tumult 
Aerial mycelium green. 
al. Growth green. 
196. Streptomyces prasinus 
b!. Growth colorless. 
121. Streptomyces hirsutus 
c!. Growth red. 
195. Streptomyces prasino- 
pilosus 
Aerial mycelium limited, produced 
late; white with tinge of gray. 
97. Streptomyces gardnert 


Soluble pigment blue or purple. 


CLASSIFICATION OF STREPTOMYCES SPECIES 157 


a. Aerial mycelium white. 
63. Streptomyces cyanofla- 
pus 
b. Aerial mycelium white to gray. 
al. Soluble pigment produced only 
on potato and certain other 
media; pigment changes to red 
in an acid and to green in an 
alkaline reaction. 
58. Streptomyces coelicolor 
b!. Pigment produced on all media, 
red in an acid and blue in an 
alkaline reaction. 
240. Streptomyces violaceo- 
ruber 
c!, Pigment at first yellow-red, 
changing to blue or bluish-green. 
190. Streptomyces plurt- 
color 
d'. Pigment purple. 
171. Streptomyces novaecae- 
sareae 
e!. Soluble pigment bluish to black. 
239. Streptomyces violaceo- 
niger 
e. Aerial mycelium blue. 
al. No spirals formed. 
36. Streptomyces caeruleus 
bt. Open spirals. 
241. Streptomyces violaceus 
3. Pigment at first green, becoming 
brown. 
a. Aerial mycelium usually absent. 
237. Streptomyces verne 
b. Aerial mycelium white. 
221. Streptomyces samp- 
sonit 
ce. Aerial mycelium brownish-white to 
brownish-gray. 
187. Streptomyces phaeovi- 
ridis 
d. Aerial mycelium dark gray, olive- 
colored, or grayish-green; sporo- 
phores produce spirals. 
244. Streptomyces viridans 
4. Pigment yellow to golden yellow. 
a. Growth green to greenish-yellow. 
al. Aerial mycelium weakly  de- 
veloped; white or pale yellow. 
242. Streptomyces virgatus 
b!. Aerial mycelium gray to dark 
gray. 
125. Streptomyces interme- 
dius 
c!. Aerial mycelium scant, white. 


Cc. 


d. 


e. 


144. Streptomyces macula- 
tus 
d'. Aerial mycelium — cinnamon- 
colored. 
158. Streptomyces murinus 


. Growth green to dark green. 


14. Streptomyces alboviri- 
dis 
Growth sulfur-yellow. 
at. Aerial mycelium white to pink- 
ish. 
81. Streptomyces flaveolus 
bt. Aerial mycelium light yellow. 
182. Streptomyces parvus 
c!. Aerial mycelium white to gray 
to reddish-gray. 
251. Streptomyces  xantho- 
phaeus 
d'. Aerial mycelium light gray. 
48. Streptomyces cellulosae 
e'. Aerial mycelium ash-gray. 
181. Streptomyces parvullus 
f'. Aerial mycelium yellowish-green 
to sulfur-yellow. 
230. Streptomyces  sulphu- 
reus 
Growth carmine red, reddish-brown 
to orange-colored to cinnamon-drab. 
al. Aerial mycelium chalk-white. 
166. Streptomyces niveoru- 
ber 
b!. Aerial mycelium white to gray. 
a®. No aerial mycelium on _ po- 
tato. 
208. Streptomyces rimosus 
b?. Aerial mycelium on potato 
white to gray to black. 
25. Streptomyces aureofa- 


clens 
Closely related form. 
222. Streptomyces saya- 
Maensis 


c?. Aerial mycelium on potato 
olive-buff. 
57. Streptomyces clavifer 
c!. Aerial mycelium grayish-brown. 
220. Streptomyces saha- 
chirot 
d'. Aerial mycelium yellowish-gray. 
110. Streptomyces griseofla- 
vus 
Growth cream-colored to brown. 
al. Rapid liquefaction of gelatin. 
7. Streptomyces albidofla- 


vUuS 


158 THE ACTINOMYCE? 


bt. Gelatin slowly liquefied. 
50. Streptomyces chibaen- 
sis 
5. Soluble pigment yellowish to yellow- 
green. 
a. Aerial mycelium white. 
51. Streptomyces 
mallus 
b. Aerial mycelium white to yellow. 
al. Growth yellowish to green. 
134. Streptomyces liesker 
bt. Growth yellow, becoming black. 
135. Streptomyces limosus 
ce. Aerial mycelium gray. 
al. Growth on sucrose nitrate agar 
yellowish-green. 
85. Streptomyces — flavovi- 


chryso- 


rens 
b!. Growth on sucrose nitrate agar 
yellow. 
47. Streptomyces — cellulo- 
flavus 


6. Soluble pigment yellowish-brown to 
reddish-brown. 
a. Growth cream-colored. 
al. Sporophores flexible and hooked. 
112. Streptomyces griseolu- 
teus 
b!. Sporophores produced in clus- 
ters. 
165. Streptomyces nitrospo- 
reus 
b. Growth has rosy tinge. 
204. Streptomyces ramnait 
ce. Growth yellowish. 
17. Streptomyces ambofa- 
clens 
d. Growth has reddish tone. 
191. Streptomyces pluricolor- 
escens 
e. Growth becoming red. 
al. Aerial mycelium white. 
70. Streptomyces eryth- 
raeus 
b'. Aerial mycelium white to gray 
with greenish tinge. 
205. Streptomyces ramulo- 
sus 
c'. Aerial mycelium mouse-gray to 
drab. 
83. Streptomyces flavogri- 
seus 
d'. Aerial mycelium white to gray 
to olive-buff. 
224. Streptomyces setonii 


oe) 


TES, Vol. II 


Soluble pigment in synthetic media 
brown. 
a. Growth coral-red. 
24. Streptomyces aurantia- 
cus 
b. Growth yellow. 
32. Streptomyces 
pensis 
c. Growth yellowish-brown 
3. Streptomyces achromo- 
genes 
d. Growth brown to purplish. 
16. Streptomyces althioti- 
cus 
e. Growth black. 
162. Streptomyces niger. 
Soluble pigment on potato plug brown 
to brownish-red to reddish-purple. 
a. Growth on potato greenish-colored. 
al. Spirals formed. 
65. Streptomyces diastati- 
cus 
b!. No spirals. 
142. Streptomyces lydicus 
b. Growth on potato gray; no spirals 
formed. 


bottro- 


42. Streptomyces canescus 
c. Growth on potato yellowish-colored. 
al. Aerial mycelium white. 
87. Streptomyces flocculus 
b!. Aerial mycelium gray to yellow- 
ish. 
a®. Growth cream-colored. 
80. Streptomyces — fimica- 
rlUus 
b?. Growth yellow-brown. 
75. Streptomyces felleus 
d. Growth on potato yellow turning 
white. 
147. Streptomyces marinoli- 
MOSUS 
e. Growth on potato pink to reddish- 
purple. 
al. Sporophores produce spirals. 
a2. Aerial mycelium cinnamon 
to drab-gray. 
170. Streptomyces nourser 
b?. Aerial mycelium gray. 
11. Streptomyces  albogri- 
seolus 
b!. Sporophores both straight and 
spiral-forming. 
228. Streptomyces spiralis 
c!. Growth on various media yellow- 
orange to brown. 
89. Streptomyces fragilis 


oP 


10. 


CLASSIFICATION OF STREPTOMYCES SPECIES 159 


Soluble pigment on synthetic agar 
brown to black. 
a. Growth on potato gray to brown. 
74. Streptomyces  exfolia- 
lis 
b. Growth on potato greenish to black. 
100. Streptomyces gelaticus 
c. Aerial mycelium pigmented green. 
101. Streptomyces glaucus 
No soluble pigment on synthetic media. 
a. Growth yellowish-brown. 
160. Streptomyces narbo- 
nensis 
b. Growth yellowish to pink to black. 
al. Aerial mycelium abundant, gray. 
23. Streptomyces armilla- 
tus 
bl. Aerial mycelium white, with 
pinkish to orange tinge on cer- 
tain media. 
77. Streptomyces filamen- 
tosus 
e'. Aerial mycelium white to yellow. 
127. Streptomyces kanamy- 
celicus 
d'. Aerial mycelium white to pink. 
145. Streptomyces madurae 
e!. Aerial mycelium scant, white. 
1838. Streptomyces pelletiert 
f'. Aerial mycelium white-gray to 
black. 
225. Streptomyces somalien- 
sis 
' Aerial mycelium black. 
180. Streptomyces paraguay- 


ge 


ensts 
ce. Growth yellowish to orange. 
al. Aerial mycelium white to rose- 
colored. 
213. Streptomyces roseofla- 
vus 
b!. Aerial mycelium white. 
201. Streptomyces putrificus 
c!. Aerial mycelium scant, white to 
grayish-brown. 
90. Streptomyces fulvisst- 


mus 
d'. Aerial mycelium orange to pale 
pink. 
226. Streptomyces spectabi- 
lis 
e!, Aerial mycelium yellowish to 
gray. 


86. Streptomyces flavus 
f!. Aerial mycelium has olive tinge. 
122. Streptomyces hominis 


d. 


g!. Aerial mycelium scant, rose- 
vellow. 
155. Streptomyces microfla- 
vUS 
h!. Aerial mycelium white to orange- 
colored. 
216. Streptomyces ruber 
i'. Aerial mycelium gray to mouse- 
gray. 
34. Streptomyces cacaoi 
Growth yellowish-green to citron- 
yellow; aerial mycelium white to 
yellow to pinkish. 
56. Streptomyces citreus 
Growth colorless to cream-colored. 
al. Aerial mycelium scant, white. 
a?. Good growth on milk. 
104. Streptomyces gougero- 
tia 
b?. No growth on milk. 
113. Streptomyces — griseo- 
planus 
b!. Aerial mycelium white to olive- 
buff. 
193. Streptomyces praecox 
e!. Aerial mycelium white. 
a®. Acid-sensitive. 
8. Streptomyces albidus 
b?. Acid-resistant. 
5. Streptomyces acidophi- 
lus 
d'. Aerial mycelium white to gray. 
a®. Sporophores straight. 
249. Streptomyces wedmor 
ensis 
b?. Sporophores produce spirals. 
197. Streptomyces pseudo 
griseolus 
e'. Aerial mycelium sandy lavender 
to dark gray. 
209. Streptomyces rochet 
f'. Aerial mycelium rose-colored. 
215. Streptomyces roseus 
Growth black. 
99. Streptomyces gedanen- 
sts 
Growth yellow to olive-ocher. 
174. Streptomyces olivaceus 


. Growth colorless to yellowish to 


olive-buff. Aerial mycelium water- 
green. 
al. Green and yellow pigments on 
malate and succinate media. 
116. Streptomyces griseus 


160 THE ACTINOMYCETES, Vol. II 


b!. No green and yellow pigments 
on malate and succinate media. 
106. Streptomyces griseinus 
i. Growth red or purple. 
39. Streptomyces californi- 
CUS 
j. Growth colorless to black. 
al. Aerial mycelium white to brown- 
ish-gray. 
202. Streptomyces pyrido- 
myceticus 
b!. Aerial mycelium on synthetic 
media dull gray. 
157. Streptomyces mita- 
kaensis 
k. Growth dark brown. 
al. Sporophores produce spirals. 
a2. Aerial mycelium white to 
gray. 
a®. Growth on potato has 
green tinge. 
118. Streptomyces halstedii 
b*. No green tinge on potato. 
219. Streptomyces  rutgers- 
ensts 
b?. Aerial mycelium olive-gray. 
169. Streptomyces nodosus 
b!. Sporophores straight. 
a2, Aerial mycelium gray-white. 
91. Streptomyces fumosus 
b?. Aerial mycelium dark gray. 
136. Streptomyces lipmanti 
1. Growth on synthetic media rose to 
gray. 
212. Streptomyces roseodia- 
staticus 
m. Growth cream-colored to yellow or 
vellow-orange. 
al. Aerial mycelium on certain me- 
dia white, moist with dark, 
glistening patches. 
124. Streptomyces hygrosco- 
picus 
bt. Aerial mycelium white-yellow 
to brownish-yellow. 
138. Streptomyces — longis- 
poroflavus 
c'. Aerial mycelium white. 
a®. Aerial mycelium present on 
protein media. 
41. Streptomyces candidus 
b?. Aerial mycelium absent on 
protein media. 
178. Streptomyces omiyaen- 


Svs 


B: 


q. 


d'. Aerial mycelium powdery white, 
with vellow tinge. 
a®. Little spiral formation. 
10. Streptomyces albofla- 
vus 
. Abundant spiral formation. 
43. Streptomyces canus 
e!. Aerial mycelium gray. 
20. Streptomyces antimy- 
colicus 


b2 


~ 
t 


. Growth colorless to pinkish to 


brown. 
159. Streptomyces naga- 
nishit 
Growth orange or red. 
al. Growth yellowish to orange; 
aerial mycelium seashell-pink. 
a®. Produces antibacterial (neo- 
mycin) and antifungal (fra- 
dicin) antibiotics. 
88. Streptomyces fradiae 
b?. Produces antiviral (luridin) 
agent. 
140. Streptomyces luridus 
b!. Growth rose to red; aerial my- 
celium white. 
a2. Growth yellow to red; weak 
proteolysis. 
13. Streptomyces albospor- 
eus 
2. Growth pale pinkish-buff; 
strong proteolysis. 
33. Streptomyces brasilien- 
Sis 
c!. Growth pale rose to red; aerial 
mycelium weakly developed, 
velvety, rose-white. 
173. Streptomyces otdio- 
sporus 
d'. Growth red; aerial mycelium 
black. 


152. Streptomyces melano- 


~ 
b 


cyclus 

Growth colorless, turning dark. 

69. Streptomyces endus 
Growth  beeoming — salmon-pink; 
acid-sensitive. 

217. Streptomyces rubescens 
Growth green to dark green; aerial 
mycelium whitish to grayish. 

245. Streptomyces viridis 
Growth on blood agar brick-red. 

164. Streptomyces — nitrifi- 


cans 


CLASSIFICATION OF STREPTOMYCES SPECIES 16] 


II. Proteinaceous media are pigmented deep 


brown to black; melanin-positive. 
1. Growth colorless on synthetic media. 
a. Aerial mycelium thin, rose-colored. 
al. Spirals produced. 
210. Streptomyces roseo- 
chromogenes 
bt. No spirals formed. 
54. Streptomyces cinnamon- 
ensis 
b. Aerial mycelium white with pale 
pink or pale gray tinge. 
131. Streptomyces — kitasa- 
waensis 
ec. Aerial mycelium gray to brown to 
reddish. 
al. Growth on organic media green- 
ish to black. 
175. Streptomyces olivochro- 
mogenes 
bt. Growth dark brown. 
206. Streptomyces — resisto- 
myctficus 
c!. Growth cream-colored. 
163. Streptomyces nigrifa- 
clens 
d. Aerial mycelium cottony, dark 
brown. 
66. Streptomyces diastato- 
chromogenes 
e. Aerial mycelium pale yellow to gray. 
30. Streptomyces blastmy- 
ceticus 


bho 
7~ 


‘owth on synthetic media yellow. 
a. Aerial mycelium white. 
203. Streptomyces rameus 
b. Aerial mycelium white to gray. 
82. Streptomyces flavochro- 
mogenes 
c. Aerial mycelium white to yellow. 
46. Streptomyces cavouren- 
SIS 
d. Aerial mycelium ash-gray. 
188. Streptomyces pilosus 
e. Aerial mycelium mouse-gray to 
green-gray. 
94. Streptomyces galbus 
f. Aerial mycelium hazel brown. 
139. Streptomyces lucensis 
g. Aerial mycelium olive-buff. 
al. Soluble pigment green to olive 
to black. 
233. Streptomyces tenuis 


bt. Soluble pigment cream-colored 
to golden brown. 
146. Streptomyces margina- 
tus 
h. Aerial mycelium white with patches 
of bluish-green on starch media. 
126. Streptomyces ipomoeae 
3. Growth white to gray. 
a. Sporophores produce spirals. 
al. Causes potato scab. 
223. Streptomyces scabies 
b!. Does not cause potato scab. 
a’, Growth on potato gray. 
119. Streptomyces hawaii- 
ensis 
b?. Growth on potato orange- 
red. 
96. Streptomyces galtieri 
b. Sporophores straight. 
at. Aerial mycelium white to gray. 
29. Streptomyces bikinien- 
Sis 
bt. Aerial mycelium white, cottony. 
156. Streptomyces mirabilis 
c. Sporophores tend to be straight; 
spirals less marked. 
28. Streptomyces beddardii 
4. Growth cream- to brown-colored. 
a. Sporophores in clusters. 
al. Aerial mycelium on _ nutrient 
agar gray to yellowish-green. 
19. Streptomyces antibioti- 
Cus 
bt. Aerial mycelium on nutrient 
agar ash-gray. 
73. Streptomyces euryther- 
mus 
c!. Aerial mycelium on nutrient 
agar white. 
38. Streptomyces caiusiae 
b. Sporophores not in clusters. 
al. Aerial mycelium white to gray. 
109. Streptomyces — griseo- 
chromogenes 
b!. Aerial mycelium olive-gray. 
248. Streptomyces _ virido- 
genes 
c!. Aerial mycelium olive-buff to 
water-green. 
107. Streptomyces — griseo- 
brunneus 
5. Growth red to reddish-orange. 
a. Aerial mycelium white. 
200. Streptomyces purpur- 
ascens 


162 THE ACTINOMYCETES, Vol. II 


b. Aerial mycelium white to gray. 
114. Streptomyces 
ruber 


griseo- 


ce. Aerial mycelium gray. 
al. No soluble pigment on synthetic 
media. 
95. Streptomyces galilaeus 
b!. Soluble pigment on synthetic 
media light carmine. 
52. Streptomyces  cinereo- 
ruber 
d. Aerial mycelium secant; ability to 
produce such mycelium easily lost. 
31. Streptomyces bobiliae 
e. Aerial mycelium pink, with bluish- 
green spores. 
28a. Streptomyces bellus 
6. Growth white to cream-colored. 
98. Streptomyces garypha- 
lus 
Growth buff to dark brown. 
a. Aerial mycelium gray to dark olive. 
105. Streptomyces gracilis 
b. Aerial mycelium dark gray. 
103. Streptomyces globosus 
ce. Aerial mycelium white. 
64. Streptomyces cylindro- 


~I 


Sporus 
d. Aerial mycelium tan to light brown. 
115. Streptomyces griseovi- 
ridis 
Growth on synthetic agar dark green 
to olive-buff. 
a. Aerial mycelium white to ight green 
to blue. 


oo 


246. Streptomyces — virido- 
chromogenes 
b. Aerial mycelium thin, white. 
211. Streptomyces roseocit- 
reus 
ce. Aerial mycelium pale gray to blue- 
gray. 
49. Streptomyces chartreu- 
SUS 
9. Growth dark brown to black. 
a. Growth on potato orange to orange- 
red. 
al. No aerial mycelium on potato. 
200. Streptomyces purpur- 
eochromogenes 
b!. Aerial mycelium scant to none; 
light brownish-gray. 
186. Streptomyces — phaeo- 
purpureus 
c!. Aerial mycelium = on_ potato 
abundant, gray. 


10. 


IE 


12. 


3: 


198. Streptomyces purpur- 
eofuscus 
d'. Aerial mycelium on potato pow- 
dery white. 
132. Streptomyces lanatus 
b. Growth on potato brown to black. 
Aerial mycelium on synthetic agar 
white to brownish. 
at. Aerial mycelium abundant. 
a®. Spirals formed. 
185. Streptomyces — phaeo- 
chromogenes 
b?. No spirals. 
153. Streptomyces  melano- 
genes 
b!. Aerial mycelium on synthetic 
agar slight. 
168. Streptomyces —nobori- 
loensis 
Growth on synthetic media colorless 
to hght orange. 
a. Aerial mycelium gray to cinnamon- 
drab. 
26. Streptomyces aureus 
b. Aerial mycelium white to gray. 
192. Streptomyces poolensis 
c. Aerial mycelium olive-buff. 
194. Streptomyces 
cundus 
Growth on synthetic agar whitish- 
vellow to grayish-yellow. 
a. Soluble pigment light yellow. 
a!. Aerial mycelium white-gray. 
a®. Aerial mycelium on potato 


praefe- 


gray. 
231. Streptomyces tana- 
shiensis 
b?. No aerial mycelium on po- 
tato. 
-oO Y oy Pee 
78. Streptomyces filipinen- 
Sis 


bt. Aerial mycelium olive-colored. 
137. Streptomyces lotdensis 
b. Soluble pigment brown to reddish- 
brown. 
154. Streptomyces michigan- 
ensis 
Growth on synthetie agar gray to olive- 
gray. 
44. Streptomyces carnosus 
Growth on synthetic media red to 
purple. 
a. Aerial mycelium white to gray. 
71. Streptomyces erythro- 
chromogenes 
b. Aerial mycelium green. 


14. 


15. 


16. 


ile(s 


18. 


CLASSIFICATION OF STREPTOMYCES SPECIES 165 


6. Streptomyces 
LENSIS 


afghan- 


c. Aerial mycelium greenish to yellow. 
4. Streptomyces acidomy- 
ceticus 
d. Aerial mycelium chalk-white. 
59. Streptomyces collinus 
Growth colorless to cream-colored. 
a. Aerial mycelium cottony white, 
lavender to vinaceous-lavender. 
133. Streptomyces lavendu- 
lae 
b. Aerial mycelium 
lavender. 
243. Streptomyces virginiae 
ec. Aerial mycelium white to cream- 
colored. 


grayish-pink to 


172. Streptomyces odorifer 
Growth yellow to brown. Aerial myce- 
hum hght tan to pink. 
236. Streptomyces 
Growth gray to black. 
79. Streptomyces 
tus 


venezuelae 
fimbria- 


Growth colorless to stone-red. 

93. Streptomyces fuscus 
Growth blue. 

62. Streptomyces cyaneus 


B. Sporophores in aerial mycelium form verticils. 
I. Melanin-negative. 


if. 


Growth yellowish. 
a. Aerial mycelium white to pinkish. 
117. Streptomyces 
joensis 
b. Aerial mycelium greenish-yellow. 
37. Streptomyces caespito- 


hachi- 


Sus 
ce. Aerial mycelium gray. 
128. Streptomyces kentuck- 
ensis 
Growth pink to red; aerial mycelium 
pink. 
76. Streptomyces fervens 
Growth yellowish to green to brown; 
aerial mycelium white. 
149. Streptomyces mashuen- 
sis 
Growth colorless on synthetic media; 
aerial mycelium white to light cinna- 
mon. 
53. Streptomyces cinnamo- 
meus 
Growth colorless; aerial mycelium 
white. 
55. Streptomyces  circula- 
tus 


6. 


Growth colorless to gray; aerial myce- 
lium white to gray. 
150. Streptomyces matensis 


Il. Melanin-positive. 


i 


to 


Sporophores do not produce any spi- 
rals. 
a. Growth white to cream-colored. 
108. Streptomyces — griseo- 
carneus 
b. Growth colorless to yellowish. 
al. Strong proteolytic properties. 
a?. Aerial 


mycelium on agar 
media absent or white 
patches. 
151. Streptomyces medioci- 
dicus 
b?. Aerial mycelium on = agar 
media white, yellowish to 
gray. 
84. Streptomyces flavoreti- 
cult 


b!. Weak proteolytic action. 
a®. Aerial mycelium white to 
yellowish 
72. Streptomyces eurocidi- 
cus 
b?. Aerial mycelium 
pale olive-buff. 
12. Streptomyces alboniger 
ce. Growth yellowish-brown. 
al. Aerial mycelium white. 
1. Streptomyces abikoen- 
sum 
b!. Aerial mycelium 
yellowish tinge. 
234. Streptomyces thioluteus 
d. Growth dark gray to gray-green. 
al. Strongly proteolytic. 
238. Streptomyces verticilla- 


white to 


white with 


tus 
b!. Weakly proteolytic. 
177. Streptomyces olivover- 
ticillatus 
e. Growth brown. 
141. Streptomyces luteover- 
ticillatus 
Sporophores produce spirals. 
a. Aerial mycelium none or limited. 
232. Streptomyces tendae 
b. Aerial mycelium yellow to ash- 
gray. 
67. Streptomyces echinatus 
ce. Aerial mycelium white. 
9. Streptomyces  albireti- 


cult 


164 


THE ACTINOMYCETES, 


d. Aerial mycelium pale vinaceous. 
161. Streptomyces netropsis 
Sporophores straight or spiral-shaped. 
207. Streptomyces reticuli 
Verticils on secondary — branches; 
growth yellowish-red to pink. 
a. Spirals produced. 
218. Streptomyces  rubrire- 
ticult 
b. No spirals formed. 
184. Streptomyces  pentati- 
CUS 
Verticils on primary and secondary 
branches. 
a. Growth yellow to brown. 
al. Aerial mycelium grayish-white. 
130. Streptomyces — kitasa- 
toensis 


Vol. 


II 


b!. Growth brown to olive-drab. 
176. Streptomyces — olivore- 
ticult 


b. Growth pink. 


Cc. 


120. Streptomyces  hiroshi- 
mensts 
Growth red to reddish-brown. 
214. Streptomyces roseover- 
ticillatus 


Sporophores may also form tufts. 


a. 


Aerial mycelium greenish-yellow, 
turning gray. 
247. Streptomyces viridofla- 


vUS 


. Aerial mycelium white. 


229. Streptomyces spirover- 
ticillatus 


Cy hya-p tier 


8 


Description of Species of Streptomyces 


the im- 
portant, recognizable species of the genus 


Streptomyces are given in this chapter. Most 


Detailed descriptions of more 


of these organisms have been isolated from 
soils, composts, peats, and water basins; 
some have come from dust and food ma- 
terials, from plant disease lesions, and from 
diseased animals and humans. Those iso- 
lated from plant disease lesions may or may 
not be the causative agents of such diseases; 
they certainly should be considered on a par 
with the soil-inhabiting forms. In the great 
majority of cases, the cultures isolated from 
diseased animals or from human infections 
as well cannot be considered as the causative 
agents of such diseases, since their patho- 
genic nature has not been established ex- 
perimentally. 

These descriptions vary greatly both in 
the details of the observations reported and 
in the uniformity of treatment of such ob- 
servations. For many of these observations, 
the author had to depend on other com- 
pilers of the literature, notably Brumpt 
(1939), Lehmann and Neumann (1927), 
Dodge (1935), Krassilnikov (1949), Erikson 
(1935), Ettlinger et al. (1958), and others. 
Unfortunately, one cannot avoid criticizing 
the tendency of certain compilers to de- 
scribe new species, and place others, often 
well recognized and previously described 
forms, as subspecies or as ‘‘also belonging 
to this species,” or the even worse tendency 
of some Classifiers to make certain minor 
variations the basis for establishing varie- 
ties of described organisms. In only 
cases was an attempt made to compare newly 


a few 


165 


isolated cultures with previously known, 
although unfortunately not always avail- 
able, type cultures. 

Although many of the synonyms have 
been examined, no detailed data are pre- 
sented literature 
Additional information can be found in the 
latest edition of Bergey’s Manual or in the 
original papers in which the descriptions 


concerning references. 


have appeared. 

Because of the growing interest in actino- 
mycetes as producers of antibiotics, numer- 
ous studies of these organisms have been 
made during the last 5 or 6 years. Many new 
species and numerous new varieties have 
been described. Old species have been better 
delineated. New 
have been proposed. Cooperative experi- 
ments have been carried out. All this ma- 


systems of classification 


terial has now been critically examined, and 
much additional information has been in- 
cluded. 

The last edition of Bergey’s Manual 
(1957) contains descriptions of 150 Strepto- 
The number has _ nearly 
doubled in the last 5 or 6 years, as indicated 
by the descriptions presented here. 


myces species. 


Description of Streptomyces Species 


1. Streptomyces abikoensum Umezawa. et 
al., 1951 TT and 
Fukuyama, S. Japan Med. J. 4: 331-346, 
1951; J. Antibiotics (Japan) 5: 469-476, 
1952; Okami, Y. zbid. 477-480). 

Morphology: Sporophores straight, short, 


(Umezawa, H., Tazaki, 


unbranched, bearing chains of spores. No 
spirals. Certain strains produce verticils. 


166 


Sucrose nitrate agar: Substrate growth 
yellowish-brown. Aerial mycelium — thin, 
yellowish-white. Soluble pigment yellowish- 
brown. 

Nutrient agar: Substrate growth cream- 
colored to yellow. No aerial mycelium. Solu- 
ble pigment brown. Melanin-positive. 

Gelatin: Growth cream-colored to brown- 
ish. Soluble pigment brown. Liquefaction 
crateriform. 

Milk: Growth brownish. Aerial mycelium 
scant, white. Soluble pigment yellowish- 
brown. Peptonization, but no coagulation. 

Potato: Growth wrinkled, cream-colored 
to brownish. Aerial mycelium yellowish- 
white. Soluble pigment reddish-brown. 

Starch agar: Growth cream-colored to 
yellowish. Aerial mycelium white. Hydroly- 
sis good. No soluble pigment. 

Nitrate reduction: Positive. 

Blood agar: Growth dark cream-yellow. 
Hemolysis strong. 

Ege media: Growth greenish-yellow. No 
aerial mycelium. Soluble pigment reddish to 
violet. 

Cellulose: Not decomposed. 

Carbon utilization: Glucose, maltose, and 
elycerol well utilized. Arabinose, xylose, 
rhamnose, fructose, galactose, mannitol, 
sorbitol, lactose, sucrose, raffinose, and inu- 
lin not utilized. 

H.S production: Negative (other strains 
positive). 

Tyrosinase reaction: Negative. 

Antagonistic properties: Produces an 
antiviral agent, abikoviromycin. 

Habitat: Soil in Japan. 

Remarks: Resembles S. fimicartus and 
S. purpureochromogenes. Gause et al. de- 
scribed a variety of this organism under the 
name of A. abikoensum var. spiralis. The 
above description was based upon strain 
2-1-6. 


* These designations represent the various cul- 
ture collections where the type cultures are de- 
posited. This has been elucidated in Chapter 4, 
p. 78-80. 


THE ACTINOMYCETES, Vol. II 


Type culture: IMRU* 3654. 

2. Streptomyces Nishimura 
et al., 1957 (Nishimura, Hi., Kamura, I. 
Tawara, K., Sasaki, K., Nakajima, K., 
Shimaoka, N., Okamoto, 8., Shimohira, M., 
and Isono, J. J. Antibiotics (Japan) 10A: 
205-212, 1957). 

Morphology: Sporophores long and 
straight; no spirals produced. Spores oval. 

Sucrose nitrate agar: Growth yellowish- 
brown. Aerial mycelium well developed, 
velvety, white. Soluble pigment dark yellow- 
ish-brown. 

Glucose-asparagine agar: Growth grayish- 
olive, thin, flat; reverse pale olive. Aerial 
mycelium velvety, almost white, slightly 
erayish. Soluble pigment at first dull yellow, 
later becoming yellowish-brown. 


aburaviensis 


Starch agar: Growth — grayish-yellow- 
brown. Aerial mycelium grayish-white. 


Soluble pigment pale yellow-brown. Hy- 
drolysis weak. 

Calcium malate agar: Growth pale yel- 
lowish-brown. Aerial mycelium thin, white 
to grayish-white. Soluble pigment grayish- 
yellow-brown. 

Nutrient agar: Growth thin, light gray. 
No aerial mycelium. No soluble pigment. 

Milk: Growth grayish-white. Aerial my- 
celium white. Coagulation and peptoniza- 
tion. 

Potato: Growth dull yellow to pale olive. 
Aerial mycelium white to light gray. No 
soluble pigment. 

Gelatin: Positive liquefaction. No soluble 
pigment. Melanin-negative. 

Nitrate reduction: Positive. 

Carbon utilization: Glycerol, dextrin, 
starch, glucose, maltose, galactose, inulin, 
and fructose utilized. Mannitol, arabinose, 
inositol, xylose, and 


rafinose, a-lactose, 


sucrose not utilized. 

3. Streptomyces achromogenes Okami and 
Umezawa, 1953 (Umezawa, H., Takeuchi, 
T., Okami, Y., and Tazaki, T. Japan. J: 
Med. Sci. Biol. 6: 261-268, 1953). 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Morphology: Sporophores straight, no 
spirals. Spores cylindrical. 

Glycerol nitrate agar: Growth colorless to 
brownish. Aerial mycelium scant, white to 
dark grayish. Soluble pigment brown. 

Glucose-asparagine agar: Growth yellow- 
ish brown. Aerial mycelium scant, yellowish- 
white. Soluble pigment none or slightly 
brown. 

Nutrient agar: Growth wrinkled, elevated, 
colorless to brownish. No aerial mycelium 
or soluble pigment. 

Potato: Growth — yellowish-brown — to 
brownish, fine, wrinkled. Aerial mycelium 
white, powdery. Soluble pigment absent at 
first, later reddish-brown. 

Gelatin: Growth yellowish-brown. Soluble 
pigment shghtly brown. Liquefaction very 
weak. Melanin-negative. 

Milk: Surface growth poor. No soluble pig- 
ment. Coagulation and slow peptonization. 

Egg media: Growth — reddish-brown, 
wrinkled. No aerial mycelium. No soluble 
pigment. 

Nitrate reduction: Positive. 

Antagonistic properties: Produces an anti- 
viral agent, achromoviromycin. 

Remarks: This culture resembles S. 
diastaticus and S. fimicarius. It is character- 
ized by the brown pigmentation on syn- 
thetic agar only. A strain of this organism, 
which produces the antibiotic streptozotocin, 
was isolated by Vavra et al. (1959) from a 
soil in Kansas; they have further cultural 
data concerning the original culture and the 
new strain. 

Type culture: IMRU 3730; ATCC 12,767. 

4. Streptomyces acidomyceticus Ogata et al., 
1954 (Ogata, K., Miyake, A., and Morimoto, 
A. Japanese Patent No. 204,403, March 5, 
1954). 

Morphology: Sporophores usually do not 
form spirals. Spores cylindrical or oval, 
0.8 to 1.2 by 1.4 to 1.8 u. 

Sucrose first 
ereenish-brown. 


nitrate agar: Growth at 


light yellow, later dark 


167 


Aerial mycelium greenish to yellow-white. 
Soluble pigment — slightly 
sometimes absent. 


violet-colored: 


Glucose-asparagine agar: Growth brown- 
ish-yellow or brownish-red, and_ partially 
greenish-blue. 

Calcium malate agar: Aerial mycelium 
Soluble 
sometimes absent. 

Glucose nutrient agar: Growth brownish, 
partially dark blue. Aerial hyphae gray- 
white. Soluble pigment brownish-black. 

Gelatin: Growth dark green. Aerial my- 
celium  greenish-white. Soluble pigment 
greenish-brown. Liquefaction limited. 

Potato: Growth greenish-brown. Aerial 
mycelium at first white, later pinkish-red. 
Soluble pigment dark green. 

Milk: Growth cream-colored, later turning 
light brown. No coagulation. Soluble pig- 
ment light brown. 

Starch: Slow decomposition. 

Tyrosinase reaction: Negative. 

Nitrate reduction: Positive. 

Production of H.S: Positive. 

Carbon utilization: Arabinose, glucose, 
maltose, lactose, salicin, and salts of or- 
ganic acids utilized. Xylose, fructose, raffi- 
nose, inulin not attacked. 

Antagonistic properties: 
antibiotic acidomycin. 

Remarks: S. acidomyceticus is closely re- 
lated to S. phaeochromogenes, the latter 


ereenish-white. pigment violet; 


Produces the 


forming spirals in gelatin media, but not the 
former. 

Type culture: ATCC 11,611. 

5. Streptomyces acidophilus (Jensen, 1928) 
Waksman and Henrici, 1948 (Jensen, H. L. 
Soil Sci. 25: 226, 1928). 

Morphology: Sporophores either few or 
numerous, with sinistrorse spirals. Spores 
oval and spherical, 1.0 to 1.2 by 1.2 to 1.5 
LL. 

Agar media: Growth on acid media (pH 
2.0 to 6.0) colorless. Aerial mycelium whitish. 

Sucrose nitrate agar: No growth. 


168 


Glucose-asparagine agar: Growth raised, 
somewhat wrinkled, young 
cultures. Aerial mycelium thin, white at 


colorless in 


first, later gray or yellowish-brown. 

Nutrient agar: No growth. 

Starch agar: Growth at 25°C good, color- 
less. Aerial mycelium abundant, smooth, 
white. Some diastatic action. 

Potato: Growth good, raised, folded. No 
discoloration of plug. Melanin-negative. 

Gelatin: Growth after 10 days very scant, 
thin, semitransparent, colorless. Liquefaction 
slow. 

Milk: No growth. 

Nitrate reduction: Trace. 

Sucrose: No inversion. 

Antagonistic properties: Strongly positive. 

Habitat: Soil. 

Remarks: Grows in acid media only, with 
an optimum at pH 3.5 to 4.5. 

6. Streptomyces afghaniensis Shimo et al. 
1959 (Shimo, M., Shiga, T., Tomosugi, T., 
and Kamoi, I. J. Antibiotics (Japan) 12A: 1, 
1959). 

Morphology: Sporophores form spirals. 
nitrate agar: Growth — olive- 
colored, with reddish-brown reverse. Aerial 
mycelium pale green to light greenish-gray. 
Soluble pigment brown to reddish-brown. 


Sucrose 


Glucose-asparagine agar: Growth olive- 
colored, with reddish-brown reverse. Aerial 
mycelium pale green to light greenish-gray. 
Soluble pigment brown to reddish-brown. 

Calcium malate agar: Growth olive- 
colored. Aerial mycelium pale yellow-orange 
to pale orange. Soluble pigment yellowish- 
brown to reddish-brown. 

Nutrient agar: Growth colorless to olive 
to buff. Aerial mycelium grayish-white. 
Soluble pigment light brown. 


Gelatin: Growth colorless. Aerial my- 
celium white. Soluble pigment brown. 


Medium liquefaction. 

Milk: Growth yellowish-brown. No aerial 
mycelium. Soluble pigment brown to dark 
brown. 


THE ACTINOMYCETES, Vol. II 


Potato: Growth wrinkled, colorless. Aerial 
mycehum olive to yellowish-brown. Soluble 
pigment yellowish-brown. 

Cellulose: Positive growth. 

Nitrate reduction: Negative. 

Tyrosinase: Doubtful. 

Carbon source: Utilizes rhamnose, raffi- 
nose, and other carbohydrates; does not 
utilize sodium citrate and sodium acetate; 
doubtful growth on dulcitol and sorbitol. 

Antagonistic properties: Produces an 
antibiotic, taitomycin, active upon gram- 
positive bacteria. 

Habitat: Soil in Afghanistan. 

Remarks: Resembles S. collinus and S. 
erythrochromogenes. 


7. Streptomyces albidoflavus (Rossi-Doria, 
1891, emend. Gasperini, 1894) Waksman and 
Henrici, 1948 (Rossi-Doria, T. Ann. ist. ig. 
sper. Roma, n. s. 1: 399-438, 1894). 

Synonym: Actinomyces albido-flavus 
Duché, 1934, emend. Krassilnikov, 1949. 

Morphology: Sporophores short, spiral- 
forming, sinistrorse. Spores spherical. 

Glucose-asparagine agar: Growth brown. 
Aerial mycelium white, later becoming 
whitish-vellow. Soluble pigment yellowish. 
agar: Growth cream- 
colored, covered with fine aerial 
mycelium; yellow soluble pigment. 

Tyrosine agar: Growth fine with orange- 
yellow on reverse side; medium becomes 


Glucose-peptone 
white 


vellowish to yellowish-rose. 

Gelatin: Punctiform colonies with white 
aerial mycelium on surface. No soluble 
pigment. Rapid liquefaction. 

Milk: Growth rapid, becoming covered 
with whitish aerial mycelium; never fully 
covering the surface; no coagulation; pep- 
tonization begins slowly and is completed in 
13 days; liquid colored yellowish-orange. 

Starch media: Growth 
covered with yellow aerial mycelium. After 
20. days, growth becomes much _ folded; 
greenish on reverse side; soluble pigment 


cream-colored, 


shghtly amber. Hydrolysis. 


DESCRIPTION OF 


Cellulose: Some growth. 

Coagulated serum: Cream-colored growth 
on surface. Aerial mycelium white. Liquefac- 
tion rapid. 

Production of HS: Negative. 

Antagonistic properties: Produces strep- 
tothricin. 

Habitat: Soil. 

Remarks: According to Flaig and Kutz- 
ner (1960), this culture obtained from CBS 
is S. coelicolor Miller. Ettlinger et al. (1958) 


considered that Duché’s strain of this 
organism belongs to S. griseus. 
8. Streptomyces albidus (Duché, 1934) 


Waksman (Duché, J. 
groupe albus. P. Lechevalier, Paris, 1934). 
form 


Les actinomyces du 


Morphology: Sporophores long, 
open spirals. Spores spherical to oval. 

Glucose nitrate agar: Growth colorless; 
some drops of colorless guttation. Aerial 
mycelium white. Soluble pigment yellowish. 

Peptone agar: Growth colorless. Aerial 
mycelium white; reverse slightly greenish. 
Soluble pigment brownish. 


Potato: Growth flat, colorless. Aerial 
mycelium white. No soluble pigment. 
Gelatin: Growth cream-colored. Rapid 


liquefaction. No soluble pigment. Melanin- 
negative. 

Milk: Growth cream-colored. Coagulation 
weak; peptonization rapid. Odor cheesy. 

Starch: Hydrolysis good. 

Cellulose: Growth good. 

Fats and waxes: Growth good, according 
to Krassilnikov (1949). 

Nitrate: Slow reduction to nitrate. 

Odor: Strong, earthy. 
properties: 
Krassilnikov (1949), it 
antagonistic activities. 

Remarks: Closely related to S. 
(Krassilnikov, 1949) ; differs by more delicate 
growth, by a reverse that is often yellowish- 
brown. Also related to S. microflavus, but 
differs from the form described by Krainsky 
in that its growth is never rose-yellow and 


Antagonistic According to 


possesses strong 


albus 


SPECIES OF STREPTOMYCES 


169 


that it grows abundantly on potato. Gause 
et al. (1957) described a variety of this 
organism under the name A. albidus var. 
invertens. Exttlinger et al. (1958) considered 


it as a strain of S. griseus. 

9. Streptomyces  albireticuli Nakazawa, 
1955 (Nakazawa, K. J. Agr. Chem. Soc. 
Japan 29: 644-647; 647-649, 1955). 

Morphology : the 
secondary verticils of the aerial mycelium. 
The spores are cylindrical 0.6 to 0.8 by 1.4 
LO eS a: 

Sucrose nitrate agar: Growth thin, color- 


Produces spirals in 


less; reverse pale ochraceous salmon. Aerial 
mycelium white. 

Glucose-asparagine agar: Growth colorless; 
becoming yellow. Aerial mycelium 
white, cottony, later becoming cream-colored. 


later 


Nutrient agar: Growth thin, mouse-gray. 
No aerial mycelium. Soluble pigment och- 
raceous tawny. 

Potato plug: Growth gray. Aerial my- 
celium white. Color of plug brown. 

Gelatin: Liquefaction slow. Soluble pig- 
ment black. 

Milk: Growth cream-colored. Peptoniza- 
tion slow. Soluble pigment brown after 24 
days. 

Starch: Actively diastatic. 

Nitrate reduction: Positive. 

Production of H.S: Positive. 

Cellulose: No growth. 

Antagonistic properties: Produces euro- 
cidin, an antifungal antibiotic. 


10. Streptomyces alboflavus (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 
(Waksman, 8. A. and Curtis, R. E. Soil Sci. 
1: 99-134, 1916; 8: 90, 1919). 

Morphology: Sporophores straight, branch- 
ing, with very little tendency to produce 
spirals. Spores oval-shaped. 

Sucrose nitrate Growth — glossy, 


agar: 


spreading, colorless, becoming yellowish. 


Aerial 
yellowish tinge. No soluble pigment. 


mycelium powdery, white, with 


170 


Glycerol malate agar: Growth light 
pinkish-cinnamon. Aerial mycelium late, 


white. 

Glucose-asparagine agar: Growth — re- 
stricted, much folded, cream-colored with 
sulfur-yellow surface. No aerial mycelium. 
No soluble pigment. 

Nutrient agar: Growth restricted, cream- 
colored. No aerial mycelium. No soluble 
pigment. 

Potato: Growth wrinkled, moist, cream- 
colored. 

Gelatin: Surface growth abundant, color- 
less. Aerial mycelium white or absent. No 
soluble pigment. Slow liquefaction. 

Milk: Surface ring pinkish. No coagula- 
tion; limited peptonization. 

Starch media: Growth thin, spreading, 
yellowish. No aerial mycelium. Good hy- 
drolysis of starch. 

Cellulose: Scant growth. 

Nitrate reduction: Positive. 

Production of H.S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Positive. 

Remarks: Various cultures related to this 
organism have been described under a 
variety of different names. It is sufficient to 
mention A. cremeus, A. griseoloalbus, A. 
flavidovirens, and a variety of the latter, 
fuscus, described by Gause et al. (1957). 
KrassiInikov (1949) considered it as a 
variety of A. flavus. 

Type culture: IMRU 3008. 


11. Streptomyces albogriseolus Benedict. et 
al., 1954 (Benedict, R. G., Shotwell, O. L., 
Pridham, T. G., Lindenfelser, L. A., and 
Haynes, W. C. Antibiotics & Chemotherapy 
4: 653-656, 1954). 

Morphology: Sporophores monopodially 
branched, producing short, compact spirals, 
averaging 4 to 6 turns. Spores spherical or 
oval, covered with numerous long, fine hairs 
(PL ln): 

Sucrose Aerial mycelium 


nitrate agar: 


THE ACTINOMYCETES, Vol. II 


white, becoming ash-gray, often with white 
spots. 

Starch agar: Aerial mycelium white to 
dark gray. Hydrolysis. 

Nutrient agar: Aerial mycelium white to 
ash-gray. Melanin-negative. 

Potato: Growth cretaceous to dirty gray- 
ish-white to faint pink. 

Carrot: Vegetative growth white to dirty 
cream; no aerial mycelium. Slant not dark- 
ened. 

Gelatin: Dirty white sediment. Positive 
liquefaction. Not pigmented. 

Milk: Orange-colored ring; partially pep- 
tonized at 14 days. 

Nitrate: Reduction to nitrite. 

Production of H.S: Negative. 

Temperature: Good growth at 25-41°C. 
No growth at 50°C. 

Antagonistic properties: Produces a ‘“‘neo- 
mycin complex.” 

Habitat: Soil. 

Type culture: IMRU 3698. 

12. Streptomyces alboniger Hesseltine et al., 
1954 (Hesseltine, C. W., Porter, J. N., 
Deduck, N., Hauck, M., Bohonos, N., and 
Williams, J. H. Mycologia 46: 16-23, 1954). 

Morphology: Sporophores — irregularly 
branched, erect to flexuous; no spirals. Ver- 
ticils produced. Spores catenulate, oval, 
0.8 by 1.25 yp. 

Sucrose nitrate agar: Growth poor, white. 
Aerial mycelium white to pale olive-buft. 
No soluble pigment. 

Glucose-asparagine agar: Growth black- 
ish-gray. Aerial mycelium white. Soluble 
pigment blackish-gray. 

Nutrient agar: Growth moist, smooth, 
colorless to 
black. No 
pigment. 

Starch agar: Growth good. Aerial my- 
celium white to pale olive-buff. Soluble 


yellowish, to dark brown or 


aerial mycelium. No soluble 


pigment black. Good hydrolysis. 


Potato: Growth moist, yellow. Aerial 


DESCRIPTION OF SPECIES OF STREPTOMYCES 171 


mycelium white. Soluble pigment dark, 
greenish-black. 

Gelatin: Growth fair. Aerial mycelium 
white. Soluble pigment light yellow. Lique- 
faction medium. 

Milk: Surface white ring, with yellow- 
green to light yellow-brown below surface. 
Aerial mycelium white. Peptonization slow. 

Cellulose: No growth. 

Production of HoS: Negative. 

Antagonistic properties: Produces puro- 
mycin, an antibiotic active upon certain 
gram-positive bacteria and protozoa. 

Habitat: Forest soil. 

Remarks: Culture is characterized by the 
formation of an olivaceous black 
pigment in some media, such as asparagine- 


soluble 


glucose agar, but no such pigment is pro- 
duced on certain organic media. 

Type culture: ATCC 12,461. 

13. Streptomyces albosporeus  (Krainsky, 
1914) Waksman and Henrici, 1948 (Krain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
II., 41: 687, 1914: Waksman, S. A. and 
Curtis, R. E. Soil Sci. 1: 99, 1916; 8: 90, 
1919). 

Morphology : straight, 
branching, with occasional spirals. Spores 
spherical or oval, 0.8 to 1.2 by 1.0 to L.8u. 

Sucrose nitrate agar: Growth spreading, 


Sporophores 


colorless, with pink center, becoming brown- 
ish, Aerial mycelium white, 
covering the whole surface; often none. No 


vinaceous. 


soluble pigment. 

Glycerol malate agar: Growth rose to 
orange-red. Aerial mycelium white, later 
changing to yellow. No soluble pigment. 

Glucose-asparagine agar: Growth wrinkled, 
spreading, red, with colorless margin. Aerial 
mycelium appears late, white. 

Nutrient agar: Small, cream-colored col- 


omies. No aerial mycelium. No soluble 
pigment. 

Starch agar: Growth thin, spreading, 
transparent, with red tinge. No aerial 


mycelium. Ready hydrolysis. 


Potato: Growth red to brownish-gray. 
No aerial mycelium, or Melanin- 
negative. 


white. 


Gelatin: Growth yellow, changing to red, 
with hyaline margin. Usually no aerial my- 
celium; when produced, sometimes gray. 
Medium liquefaction. 

Milk: Seant, pink ring. No coagulation; 
no peptonization. 

Cellulose: No growth or scant. 

Nitrate reduction: Fair. 

Production of H.S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Positive. 

Habitat: Soil. 

Remarks: Above description is based 
partly upon the isolates of Waksman and 
Curtis, since Krainsky’s culture was not 
available. Krassilnikov (1949) considered it 
as a variety of A. ruber. According to 
Ettlinger et al. (1958) this organism should 
be considered as a strain of S. griseus, a 
hardly justifiable assumption. 

Type culture: IMRU 3003. 


14. Streptomyces alboviridis (Duché, 1934) 
Waksman (Duché, J. Les actinomyces du 
groupe albus. P. Lechevalier, Paris, p. 317, 
1934). 

Morphology: According to Krassilnikov 
(1949), the sporophores produce spirals with 
3 to + turns. Spores spherical. 
nitrate Growth 


Glucose agar: 


colored becoming olive-green. Aerial myce- 


cream- 


lum white, becoming yvellowish-green. Solu- 
ble pigment brownish. 

Glucose-asparagine agar: Growth at first 
white, becoming olive-colored to almost 
dark. Aerial mycelium white to green. Solu- 
ble pigment yellowish. 

Starch agar: Growth cream-colored; re- 
verse brownish-green. Aerial mycelium 
white, becoming green. 

Gelatin: Growth white, becoming green. 
Soluble pigment greenish-brown. Liquefac- 
tion rapid (slow, according to Krassilnikov, 


1949). 


172 THE ACTINOMYCETES, Vol. II 


Potato: Growth white, becoming brown- 
ish to rust-colored. Plug colored black. 

Tyrosine agar: Growth white with a 
brownish reverse. Soluble pigment brownish. 

Milk: Coagulation and peptonization. 

Coagulated serum: Growth cream-colored. 
No aerial mycelium. No soluble pigment. 
Rapid liquefaction of serum. 

Remarks: This organism is considered as 
a transitional form between S. albus and S. 
viridis. Krassilnikov (1949) considered it as 
a variety of A. viridochromogenes. 


15. Streptomyces albus (Rossi-Doria, 1891; 
emend. Gasperini, 1892) Waksman and Hen- 
rici, 1948 (Rossi-Doria, T. Ann. ist. ig. sper. 
Roma, n. s. 1: 399-438, 1894). 

Synonym: Numerous synonyms of this 
species are found in the literature. They 
belong mostly to the species-group ““S. albus.” 
Many of them are listed in Chapter 6, under 
the corresponding group. 

Morphology: Sporophores produce long 
spirals. Spores spherical to oval. Some strains 
produce, according to Okami, straight sporo- 
phores, depending on the composition of the 
medium. 

Agar media: Growth colorless; may be- 
come yellowish to brown with age. No 
pigment formed, although 
strains may excrete a brownish substance in 
certain media and under certain conditions. 
Aerial mycelium abundant, white; the shade 
of color varies with composition of medium 


soluble some 


from snow-white to somewhat yellowish. 

Sucrose nitrate agar: Substrate growth 
smooth, colorless. Aerial mycelium cottony 
to powdery; white to snow-white. 

Glucose-asparagine agar: Aerial mycelium 
gray, becoming brownish. 

Nutrient agar: Generally no aerial my- 
celium; chalky white deposit on old colonies. 

Potato: Growth lichenoid, cream-colored. 
Aerial mycelium white. 

Gelatin: Colonies gray. No soluble pig- 
ment. Strong liquefaction. 


Milk: Surface ring cream-colored. Aerial 
mycelium white. Peptonization rapid. 

Starch agar: Aerial mycelium white. 
Rapid hydrolysis of starch in some cultures; 
others show little or no hydrolysis. 

Nitrate: Reduction to nitrite positive. 

Production of H.S: Negative. 

Odor: Characteristic, moldy. 

Antagonistic properties: Certain strains 
are active upon gram-positive bacteria. Some 
produce actinomycetin, others form thiolutin 
or endomycin. 

Habitat: Occurs in dust and soil. 

Xemarks: The general occurrence of this 
species, the ease of its superficial identifica- 
tion, and the fact that it has been adopted 
as the type species for the genus Strepto- 
myces, Justify a more complete characteri- 
zation, as given in Chapter 6. Numerous 
strains of this species, varying in. their 
cultural and other properties have been 
reported. Numerous descriptions of closely 
related organisms also are found in the 
literature (Duché). IXrassilnikov lists 18 
strains and substrains (4. albus vulgaris, A. 
albus chlamydosporus, etc.). A. longisporus 
KrassilInikov (1949) and some of the sub- 
strains, like A. longisporus griseus, belong to 
this group. Solovieva and Rudaya (Anti- 
biotiki, 4(6): 5-10, 1959) list a variety fun- 
gatus capable of producing an antifungal 
agent, albofungin. 

Type culture: IMRU 3005. 

16. Streptomyces  althioticus Yamaguchi 
et al., 1957 (Yamaguchi, H., Nakayama, 
Y., Takeda, K., Tawara, K., Maeda, K., 
Takeuchi, T., and Umezawa, H. J. Anti- 
biotics (Japan) LOA: 195-200, 1957). 

Morphology: Curved chains or spirals of 
oval spores on ends of aerial sporophores. 
Frequently, tips of aerial mycelium divided 
into tufts of spore chains. 

Sucrose nitrate agar: Growth colorless to 
white, later light brown to purplish. Aerial 
mycelium powdery white, later gray. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Glucose-asparagine agar: Growth color- 
less to white, later light brown with or 
without dull light reddish tinge. Surface 
glossy. Aerial mycelium scant, white. Light 
brown to dull light reddish-brown soluble 
pigment. 

Starch agar: Aerial mycelium white to 
eray. No soluble pigment. No hydrolysis in 
7 days. 

Glucose-asparagine agar: Growth color- 
less to white, later light brown with or with- 
out dull light reddish tinge. Surface glossy. 
Aerial mycelium scant, white. Light brown 
to dull light reddish-brown soluble pigment. 


Glucose nutrient agar: Growth heht 
yvellowish-brown. Surface glossy. Aerial 


mycelium white to gray. Soluble pigment 
light yellowish-brown. 

Potato: Growth abundant, light yellowish- 
brown. Aerial mycelium white to gray. 

Gelatin: Scant growth. No liquefaction. 
Brown soluble pigment. 

Milk: Light yellowish-brown surface ring, 
with scant white aerial mycelium. Yellow- 
ish-orange soluble pigment occasionally. 
Peptonization positive. 

Egg medium (37°C): Growth yellow with 
eray tinge. Aerial mycelium white, later 
light purplish occasionally. 

Cellulose: Scant growth with purplish- 
gray aerial mycelium and light purplish 
pigment. 

Carbon utilization: Abundant growth with 
rhamnose, fructose, galactose, mannitol, and 
glucose; weak growth with xylose, arabinose, 


maltose, sorbitol, and inositol; none = or 
very scant with dulcitol, raffinose, and 
inulin. 


Antagonistic properties: Produces anti- 
biotic althiomycin. 

Remarks: Closely related to S. achromo- 
genes and S. rimosus. Spiral formation of 
culture, no nitrite formation, and purplish 
tone of growth and aerial mycelium differ- 
entiate it from S. achromogenes. Purplish 
tinge of aerial mycelium and growth, soluble 


Lis 
pigment, no nitrite formation, and no 
cracked surface of the growth differentiate 
it from S. rimosus. 


17. Streptomyces ambofaciens Pinnert-Sin- 
dico, 1954 (Pinnert-Sindico, S. Ann. 
Pasteur 87: 703-707, 1954). 

Morphology: Sporophores form spirals. 
Spores oval or spherical. 


inst. 


Sucrose nitrate agar: Substrate growth 
yellow to gray. Aerial mycelium white to 
gray. Soluble pigment weak brownish-yel- 
low. 

Glucose-asparagine agar: Growth yellow, 
covered with white aerial mycelium. Soluble 
pigment weak yellow-brown. 

Calcium malate agar: Growth similar to 


that on sucrose nitrate agar. No soluble 
pigment. 
Potato: Growth clear brown. Aerial 


mycelium powdery gray. Soluble pigment 
weakly brown to brownish-red. 

Gelatin: Surface growth yellow; flakes in 
liquefied portion. Medium 
Weak brown-orange pigment in liquefied 
zone. Melanin-negative. 

Milk: No coagulation, partial peptoniza- 
tion in | month. Peptonized zone orange- 


liquefaction. 


brown to red. 

Nitrate: Weak 
synthetic media; none at all in organic 
media. 

Production of HoS: Negative. 

Carbon utilization: Glycerol, arabinose, 


reduction to nitrite in 


glucose, galactose, levulose, mannose, lac- 
tose, rhamnose, starch, and mannitol well 
utilized. Raffinose, erythritol, dulcitol, and 
sorbitol not utilized. 
Antagonistic properties: Produces two 
antibiotics, congocidin and spiramycin. 
Remarks: Ettlinger ef al. (1958) included 


this organism with S. auwreofaciens. 
(Beijerinck, 
1912; emend. Krassilnikov, 1941) Waksman 


18. Streptomyces annulatus 


(Krassilnikov, N. A. Actinomycetales. Izvest. 
Akad. Nauk, SSSR, Moskau, 1941). 


174 


Not A. annulatus Wollenweber, 1920. 

Morphology: Sporophores produce spirals, 
with 3 to 7 turns (sinistrorse). Spores spheri- 

= 
cal, 0:7 bm. 

Sucrose nitrate agar: Growth colorless, 
flat, penetrating deep into agar. Aerial 
mycelium white, velvety, growing in the 
form of concentric rings. 

Nutrient agar: Colorless growth. Aerial 


mycelium white, concentric rings — less 
marked. Melanin-negative. 

Gelatin: Slow hquefaction. 

Milk: Positive coagulation and slow 


peptonization. 

Starch: Hydrolysis. 

Cellulose: Growth good. 

Invertase: Positive. 

Production of HS: Negative. 

Odor: Strong, earthy. 

Antagonistic properties: Highly antag- 
onistic to mycobacteria and gram-positive 
bacteria; some strains are active against 
fungl. 

Habitat: Soil. 

Remarks: Krassilnikov (1949) 
this organism as a variety of S. albus. 

Type culture: IMRU 3307. 

19. Streptomyces antibioticus (Waksman 
and Woodruff, 1941) Waksman and Henrici, 
1948 (Waksman, 8S. A. and Woodruff, H. B. 
J. Bacteriol. 42: 232, 246, 1941; see also 
Waksman, 8. A. and Gregory, F. J. Anti- 
biotics & Chemotherapy 4: 1050-1056, 1954). 

Morphology: Sporophores straight, long, 


considers 


arranged in clusters or broom-shaped bodies; 
usually not wavy and no spirals; some 
strains may produce a few spirals. Spores 
nearly spherical to somewhat elliptical, 
smooth (Pl. II 1). Capacity to produce aerial 
mycelium lost upon continued 
cultivation on artificial media (Pl. V Fb). 
Sucrose nitrate 


may be 


agar: Growth cream- 
colored to yellowish, tending to darken in 
reverse. Aerial mycelium light to mouse- 
gray, with white patches. Soluble pigment 


faint yellowish to yellow to dark. 


THE ACTINOMYCETES, Vol. II 


Glucose-asparagine agar: Growth cream- 
colored, with yellowish to orange to dark 
reverse. Aerial mycelium light to ash-gray. 
Soluble pigment absent or yellow to brown- 
ish. 

Calcium malate agar: Growth colorless to 
yellowish. Aerial mycelium white to white- 
eray. 

Nutrient agar: Growth brownish, thin. 
Aerial mycelium yellowish-gray to yellow- 
ish-green. Soluble pigment brown to dark. 
Melanin-positive. 

Potato: Growth thin to heavy, lichenoid; 
brownish to orange in color, sometimes 
olive-green. Aerial mycelium absent or 
thin to patchy, white or gray. Soluble pig- 
ment brownish to dark; absent in many 
cultures. 

Gelatin: Growth yellowish to brown to 
dark brown. Aerial mycelium as patches of 
white to gray. Soluble pigment black. 
Liquefaction at first very slow, later be- 
coming more rapid. 

Milk: Thick surface ring, brownish. Aerial 
mycelium mouse-gray with greenish tinge. 
No coagulation, but gradual peptonization. 
Soluble pigment brownish to black. 

Production of HS: Positive. 

Tyrosinase: Negative. 

Antagonistic properties: Marked antag- 
onistic effect on bacteria and fungi. Produces 
actinomycin A, the first crystalline anti- 
biotic ever isolated from an actinomycete 
culture. 

Source: Isolated from soil on Escherichia 
coli-washed plate, using living cells of F. 
coli as the only source of available nutrients. 
Later also isolated from a variety of dif- 
ferent soils. 

Remarks: Ettlinger et al. (1958) included 
in this group S. bikiniensis, S. cinereoruber, 
S. eurythermus, and S. tpomoeae. Krassil- 
nikovy (1949) included this species with A. 
parvus. 


Type culture: IMRU 3435. 


20. Streptomyces antimycoticus Waksman 


DESCRIPTION OF SPECIES OF STREPTOMYCES 175 


(Leben, C., Stessel, G. J., Keitt, G. W. 
Mycologia 44: 159-169, 1952). 

Morphology: 
situated typically in dense groups. Spirals 
tend to be open, becoming closed and com- 
pact prior to the formation of spores. Spores 
oval, 0.6 to 1.3 by 0.7 to 2.0 yu. 

Sucrose 
at first white, later gray. Aerial mycelium 
abundant, light neutral gray. No soluble 


Sporophores with spirals 


nitrate agar: Substrate growth 


pigment. 

Glycerol malate agar: Aerial mycelium 
abundant, light neutral gray. Soluble pig- 
ment faint green. 

Nutrient peptone agar: Growth shiny, 
cream-colored. Aerial mycelium moderate, 
pebbly, white. No soluble pigment. Melanin- 
negative. 

Potato-glucose agar: Aerial 
abundant, neutral gray. Soluble pigment 


mycelium 


faint, brown. 

Yeast extract Aerial 
abundant, neutral gray. No soluble pigment. 

Starch agar: Aerial mycelium abundant, 
white to neutral gray. No soluble pigment. 
Diastatic action weak to moderate. 

Potato: Growth finely wrinkled, cream- 
colored. Aerial mycelium sparse. Plug dark- 
ened slightly. 

Gelatin: Growth — translucent, 
colored. Aerial mycelium sparse, 
Liquefaction slight at 15 days, moderate 
at 30 days. No soluble pigment. 

Milk: Ring cream-colored. Coagulation; 
peptonization in 15 to 30 days. Yellowish- 


agar: mycelium 


cream- 
white. 


orange pigmentation. 

Nitrate reduction: Shght. 

Antagonistic properties: Produces an anti- 
fungal agent, helixin. 

21. Streptomyces arenae Grundy, 1954 
(Grundy, W. E. Brit. Pat. 719,230, Dec. 1, 
1954*). 

Morphology: Monopodial branching of 
mycelium. Sporophores terminate in tight 
spirals. Spores spherical to oval. 


* Supplemented by personal communication. 


Sucrose nitrate agar: Growth wrinkled, 
vellow, turning dark orange-brown with age. 
Aerial mycelium grayish-white. Soluble pig- 
ment light yellow-brown. 

Calcium malate 
colored, turning bright reddish-brown with 


agar: Growth cream- 
age. Aerial mycelium fluffy, cream-colored 
turning gray with pink tinge. Soluble pig- 
ment light pink. Complete dissolution of the 
calcium malate. 

Glucose-asparagine agar: Growth sparse, 
golden brown; a few tufts of white aerial 
mycelium. Soluble pigment yellow. 

Nutrient agar: Growth moderate, golden 
brown. Aerial mycelium gray-white, spores 
turning darker gray. Soluble pigment light 
brown. 

Potato: Growth abundant, golden brown, 
turning dark brown. Aerial mycelium fluffy, 
becoming on sporulation dark gray. Potato 
dark gray, turning black. 

Gelatin: Heavy gray pellicle on surface. 
Liquefaction slow. Soluble pigment deep red- 
brown diffusing through the liquefied zone. 
Medium liquefaction. 

Milk: Heavy pellicle. Milk digested in 25 
to 28 days with formation of curd just before 
complete digestion. Soluble pigment dark 
brown, turning black in 30 days. 

Starch: Hydrolysis slow. 

Nitrate: No reduction. 

Carbon utilization: Good growth with 
xylose, glucose, mannose, galactose, lactose, 
maltose, sucrose, starch, mannitol, glycerol 
sodium acetate, sodium citrate, and potas- 
sium sodium tartrate. Sorbitol and calcium 
lactate not utilized. 

Antagonistic properties: Produces an anti- 
biotic active upon Mycobacterium tuberculo- 
Sts. 

Habitat: Illinois soil. 

22. Streptomyces argenteolus Perlman, 
1957 (Perlman, D. U. S. Patent 2,709,705, 
October 7, 1958). 

Morphology: Aerial 
generally branched, not forming loops or 


mycelium hyaline, 


176 


spirals; individual filaments are rarely sep- 
tate. straight, flexuous, or 
fascicled (in tufts). Spores are oval to oblong, 
1.0 to 1.2 uw. The spore color is ight gull-gray. 
Sucrose nitrate agar: No growth. 
Nutrient agar: Growth colorless, abun- 


Sporophores 


dant, spreading. Aerial mycelium white. No 
soluble pigment. 

Oatmeal agar: Growth good. Aerial myce- 
lium limited, no sporulation. Soluble pig- 
ment shght maize-yellow. 

Casein digest-meat extract agar: Growth 
abundant, dark olive-buff. Aerial mycelium 
well developed, pale smoke-gray. No soluble 
pigment. 

Gelatin: Rapid liquefaction. Melanin-neg- 
ative. 

Milk: Positive coagulation and peptoniza- 
tion. 

Potato: Growth good, creamy-buff, cere- 
briform. Aerial mycelium white; no sporula- 
tion. No soluble pigment. 

Starch: Hydrolyzed. 

Nitrate reduction: Positive. 

Carbon utilization: Mannitol, d-xylose, 
l-arabinose, /-rhamnose, d-fructose, treha- 
lose, and lactose utilized. No growth or very 
scant growth with inositol, sorbitol, meli- 
biose, sucrose, and dextrin. 

Habitat: Soil. 

Biochemical activities: Certain strains of 
this organism convert progesterone to 16 
a-hydroxyprogesterone. 


23. Streptomyces armillatus Mancy-Cour- 
tillet and Pinnert-Sindico, 1954 (Mancy- 
Courtillet, D. and Pinnert-Sindico, S. Ann. 
inst. Pasteur 87: 580-584, 1954). 

Morphology: Aerial mycelium produces 
spirals. 

Glucose- or glycerol-asparagine 
Growth yellow-gray. Aerial mycelium poorly 


agar: 


developed, white. 

Sucrose nitrate agar: Growth very poor, 
colorless. No aerial mycelium. 

Glucose nitrate agar: Growth poor. No 
soluble pigment. No reduction of nitrate. 


THE ACTINOMYCETES, Vol. II 


Glucose-peptone agar: Growth very good, 
vellow-gray. Aerial mycelium poorly devel- 
oped, white. Soluble pigment weak rose, 
becoming brownish. Melanin-negative. 

Potato: Growth good, yellow-gray. Aerial 
mycelium 
pigment. 


poorly developed. No soluble 
Tyrosine medium: Growth flat, yellow- 


gray, becoming beige. Aerial 
white. No soluble pigment. 

Gelatin: Growth in form of pellicle. Aerial] 
mycelium white. Soluble 
brown. Rapid liquefaction. 


Milk: Growth in form of surface ring, 


mycelium 


pigment rose- 


eray to yellow. Peptonized portion colored 
yellow. Coagulation and rapid peptoniza- 
tion. 

Starch: No hydrolysis. 

Antagonistic properties: Produces oxytet- 
racyclhine. 

temarks: This organism can be classified 
with the S. bobiliae-S. erythreus group, al- 
though its growth is yellow rather than red. 
It grows poorly upon synthetic media, upon 
which it forms no aerial mycelium. It does 
not reduce nitrate to nitrite. It does not 
produce a purple pigment upon egg media. 
It does not change the reaction of milk to 
alkaline. It does not hydrolyze starch. It 
differs from S. rimosus and S. griseoflavus, 
which produce yellow to brown pigments; 
S. armillatus under the same conditions does 
not form any pigment. 


24. Streptomyces aurantiacus — (Rossi- 
Doria, 1891 emend. Gasperini, 1892; emend. 
Waksman 
A. Actinomycetales. Izvest. Akad. 
SSSR, Moskau, p. 36, 1941). 


Morphology: Produces an abundance of 


(Krassilnikov, N. 
Nauk. 


Kkrassilnikov ) 


chlamydospores. Sporophores form. spirals 
with 3 to 5 turns. Spores spherical to oval, 
0.7 to 0.9 by 0.6 to 0.8 u. 

Agar media: Growth lichenoid, dry, com- 
pact; colored bright orange or golden. Pig- 
ment insoluble in medium, but soluble in 


DESCRIPTION OF SPECIES OF STREPTOMYCES 177 


organic solvents. Aerial mycelium poorly 
developed. Melanin-negative. 

Potato: Soluble pigment brown. 

Gelatin: Growth yellow to orange-yellow 
to deep orange. Liquefaction none or slow. 
No aerial mycelium. 

Milk: Surface growth orange. No coagula- 
tion; unchanged or weak peptonization. 

Starch: Slow hydrolysis. 

Cellulose: No growth. 

Nitrate: No reduction. 

Invertase: None. 

Fats: Hydrolysis and utilization rapid. 

Paraffin: Growth good, with spiral-form- 
ing sporophores and spherical spores. 

Pigment: Red-orange, extracted with 96 
per cent alcohol. The orange pigment was 
dissolved in petroleum ether, the red pig- 
ment being insoluble (Ixriss). 

Antagonistic properties: Strongly antago- 
nistic to gram-positive bacteria. 

Habitat: Soil, dust. 


Remarks: Some strains deposit ferric 
oxide on the surface of the hyphae. 
25. Streptomyces aureofaciens Duggar, 


1949 (Duggar, B. M. Ann. N. Y. Acad. Sci. 
51: 177, 1948; U.S. Patent 2,482,055, Sept. 
14, 1949). 

Morphology: Sporophores monopodially 
branched, flexuous, producing open spirals. 
Spores spherical to oval, smooth (PI. II m). 

Sucrose nitrate agar: Substrate growth 
only. Occasionally faint brownish pigment 
produced. 

Glucose-asparagine-meat 
Growth hyaline, changing to orange-yellow 
Aerial mycelium, if 


extract agar: 
or purplish-brown. 
present, white, changing to ash-gray or dark 
gray with tawny reverse. Faint yellowish 
soluble pigment occasionally discernible. 

Nutrient agar: Growth good, translucent 
to brownish. No aerial mycelium. No soluble 
pigment. Melanin-negative. 

Potato: Growth lichenoid, light orange- 
yellow to brown-red to purplish. No aerial 
mycelium. Color of plug unchanged. 


Gelatin: Cream-colored surface ring. Liq- 
uefaction none to limited. No soluble pig- 
ment. 

Milk: Growth limited, yellow-brown sur- 
face. Coagulation and peptonization variable 
(often none, occasionally present). 

Production of HS: Mostly negative. 

Antagonistic properties: Produces chlor- 
amphoteric compound 
and 
various 
viruses. 


tetracycline, an 
both 
chlorine, active against 
rickettsiae, the 
organism also produces, especially in a 
chlorine-poor medium, tetracycline. The 
presence of phosphorus in the medium in- 
fluences not only growth but also antibiotic 


containing nitrogen non-ionic 


bacteria, 
The 


and larger 


production (Prokofieva-Belgovskaya and 
Popova, 1959). 

Habitat: Soil. 

Remarks: The numerous natural and 


induced variants of S. aureofaciens display 
wide variations in color of substrate growth, 
ranging from pale yellow to reddish-brown, 
and even occasionally greenish, depending 
upon the composition of the nutrient sub- 
strates and environmental conditions (Dug- 
gar et al., 1954). Color of aerial mycelium 
is influenced by sporulation. Ettlinger e¢ al. 
(1958) included S. ambofaciens in this group. 

Type culture: IMRU 3550; ATCC 10,762. 

26. Streptomyces aureus (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 
(Waksman, 8S. A. and Curtis, R. E. Soil Sci. 
1: 24, 1916; 8: 97, 1919). 

Morphology: Aerial 
sporophores with numerous closed spirals; 


mycelium forms 
some strains produce flexible sporophores 
with open spirals. Spores spherical to oval, 
0.6, to 1.0 by 0.8 to 1.4 uw (Fig. 32). 

Sucrose nitrate agar: Growth thin, spread- 
ing, colorless, becoming dark brown. Aerial 
mycelium thin, powdery, mouse-gray, be- 
coming cinnamon-drab. No soluble pigment. 
Growth cream- 
black. Aerial 
mycelium light brown. No soluble pigment. 


Malate-glycerol 


agar: 


colored, with surface almost 


178 


Glucose-asparagine agar: Growth light 
orange; raised center, hyaline margin. Aerial 
mycelium hght drab. 

Nutrient agar: Growth restricted, gray. 
No aerial mycelium. Soluble pigment deep 
brown. 


Starch agar: Growth thin, transparent, 


, 


THE ACTINOMYCETES, Vol. II 


Aerial buff-colored. 
Good hydrolysis. 

Potato: Growth abundant, wrinkled, 
brown, becoming black. Aerial mycelium 
white to ash-gray. Soluble pigment black. 

Gelatin: Surface growth fair, cream-col- 
Aerial mycelium 


spreading. mycelium 


ored, becoming brown. 


Figure 32. Sporophores of S. aureus, X 30,000 (Courtesy of KE. Baldacci, University of Milan, Italy). 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


absent or white. Brown soluble pigment. 
Liquefaction rapid, later slowing down. 

Milk: Black ring. Limited coagulation and 
peptonization. 

Nitrate: Reduction to nitrite with certain 
‘carbon sources. 

Invertase: None to positive. 

Temperature: Optimum 25°C. 

Antagonistic properties: Produces poly- 
enes, substances active against various 
fungi. Some strains produce luteomycin. 

Habitat: Soil. 

temarks: Cultures under this name were 
described by DuBois-Severin in 1895, by 
Lachner-Sandoval in 1899, and by Sartory 
in 1923. Yamaguchi and Saburi (1955) re- 
ported that the S. aureus culture obtained 
from collections produces straight aerial 
hyphae and no spirals when grown on var- 
ious synthetic media. Okami and Suzuki 
(1958) isolated two strains that produced 
spirals. Ettlinger et al. (1958) considered 
this organism as a strain of S. griseus. IKras- 
silnikov (1949) considered it as a variety of 
A. flavus. 

Type culture: IMRU 3309. 


27. Streptomyces autotrophicus Takamiya 


and Tubaki, 1956 (Takamiya, A., and 
Tubaki, K. Arch. Mikrobiol. 25: 58-64, 
1956). 


Morphology: Sporophores alternately or 
irregularly branched, breaking up into 
spores; no spiral formation. Spores colorless 
with smooth surface; varying in shape from 
ellipsoid to long ovoid or cylindrical; usually 
2.5 to 4.8 by 0.5 to 0.8 uw, sometimes smaller, 
ib by 0:3 %u- 

Nitrate, carbohydrate-free, agar: Aerial 
mycelium powdery and snow-white in ap- 
pearance, consisting of a tough mycelial felt; 
thicker at central area than at periphery. 
Reverse side of growth wrinkled and pale 
yellowish. No soluble pigment. 

Calcium malate agar: Growth much 
folded, and raised in central area, cream- 
yellow at earlier stages of development; 


reverse side pale brownish. Aerial mycelium 
white. 

Nutrient agar: 
raised; reverse side relatively smooth 


Growth much folded and 
and 
pale brownish. Aerial mycelium snow-white. 
In old cultures, a faint brown tint in agar 
layer immediately beneath growing colony. 

Malt agar: Growth irregularly wrinkled 
and folded; reverse side wrinkled and pale 
yellowish. Production of spores rather poor. 

Starch agar: Growth scanty; no hydroly- 
sis. 

Potato: Growth colorless, much folded, 
with thick central area and thin periphery, 
pale brownish. 

Gelatin: No liquefaction. 

Milk: Thin white pellicle formed on sur- 
face. Reverse side yellowish. No coagulation. 
No soluble pigment. 

Cellulose: Not decomposed. 

Nitrate reduction: None. 

Habitat: Originally found on the surface 
of phosphate buffer solution left unused in a 
laboratory in Tokyo. Conceivably, it was 
derived from atmospheric dust. 

temarks: Hirsch (1960) considers this or- 
ganism as a Nocardia (N. autotrophica), ca- 
pable of utilizing petroleum. 


28. Streptomyces beddardii (Erikson, 1935) 
Waksman (Erikson, D. Med. Research 
Council (Brit.) Spec. Rept. Ser. 203: 13-14, 
1935). 

Morphology: Sporophores long, slender, 
forming many wavy or closely coiled spirals, 
particularly on glucose agar; spirals less 
marked or lacking on poorer nutritive media 
like synthetic glycerol agar or water agar. 
Aerial hyphae straighter and more branched 
with shorter sporophores on starch agar. 
Spores oval. 

Glucose-asparagine agar: Growth wrin- 
kled, membranous. Aerial mycelium scant, 
white. 

Nutrient agar: Growth colorless, coherent, 
wrinkled, membranous. Aerial mycelium 
scant, white. Soluble pigment deep brown. 


180 

Starch agar: Growth spreading, colorless. 
Aerial mycelium abundant, white. Hydroly- 
sis. 

Egg medium: Growth extensive, wrinkled, 
bright yellow. Considerable liquefaction. 

Blood agar: Growth in uniformly striated 
colorless bands; occasional round colonies at 
margin. Hemolysis positive. 

Potato: Growth moist, colorless. Aerial 
mycelium scant, white at top of plug. 

Gelatin: Dull white flakes sinking to bot- 
tom as medium liquefies. Rapid liquefaction. 

Milk: Coagulation followed by peptoniza- 
tion. 

Source: Human spleen in a case of splenic 
anemia. No record concerning actual patho- 
genicity. 

28a. Streptomyces bellus Margalith and 
Beretta (Margalith, P. and Beretta, G. 
Mycopathol. Mycol. Appl. 12: 189-195, 
1960). 

Morphology : 
flexuous with short, open hooks. 

Sucrose nitrate agar: Substrate growth 
light cherry-pink. Aerial mycelium pinkish- 
white. Soluble pigment light pink. 

Glucose-asparagine agar: Growth colorless 
to hight pink with orange tinge. Aerial myce- 
lium white, with small amount of bluish 
spores. Soluble pigment lght pink with 


Sporophores long, straight, 


orange tinge. 

Calcium malate agar: Substrate growth 
pink, with pinkish-violet reverse. Sporula- 
tion abundant, pinkish-blue. Soluble pig- 
ment pinkish-violet. 

Nutrient agar: Substrate growth hyaline 
with Aerial mycelium 
slight or absent. Soluble pigment brown. 

Starch agar: Growth hyaline with color- 
less to pale pink reverse. Aerial mycelium 


brownish reverse. 


limited, with bluish-green spores. Starch 
hydrolyzed. 
Glucose-casein — digest-yeast-beef 
Growth pink. Aerial mycelium pinkish with 
bluish-green spores. Soluble pigment light 


agar: 


pink. 


THE ACTINOMYCETES, Vol. II 


Glucose-yeast extract-beef-peptone agar: 
Growth pink. Aerial mycelium pinkish- 
white with bluish-green spores. Soluble pig- 
ment reddish-brown. 

Potato: Growth rough, colorless to brown. 
No aerial mycelium. Soluble pigment brown. 

Gelatin: Slow liquefaction. Soluble pig- 
ment brown. 

Milk: Growth in form of brownish ring. 

Nitrate reduction: None. 

Cellulose: Moderate growth. 

Carbon utilization: Most sugars readily 
utilized. Xylose, inulin, and dulcitol not 
utilized in solid media. Succinate, citrate and 
glycine not utilized. 

Antagonistic properties: Produces anti- 
biotic matamycin, active upon gram-positive 
bacteria. 

Habitat: Soil in Italy. 

29. Streptomyces bikiniensis Johnstone 
and Waksman, 1948 (Johnstone, D. B. and 
Waksman, 8. A. J. Bacteriol. 55: 317-326, 
1948). 

Morphology: Sporophores straight. Spores 
oval (Fig. 33). 

Sucrose nitrate agar: Growth white, be- 
coming pallid neutral gray with white tinge. 
Aerial mycelium abundant, white to gray. 
Soluble pigment light brown. Superficial 
droplets amber-colored. 

Glucose-asparagine agar: Growth abund- 
ant. Aerial mycelium white to mouse-gray. 
Soluble pigment light amber. 

Nutrient agar: Growth luxuriant. Aerial 
mycelium moderate, white. Soluble pigment 
deep brown. 

Starch agar: 
mycelium white, becoming gray. Slight hy- 


Growth abundant. Aerial 
drolysis. 

Potato: Growth wrinkled and raised, pale 
ochraceous buff. Soluble pigment brown to 


black. 
Gelatin: Shght liquefaction. 
Milk: Surface growth patchy, white. 


Aerial mycelium gray. Gradual peptoniza- 
tion. 


DESCRIPTION OF 


ay 


4i2@ 


S. bikiniensis, 


FIGURE 33. 
tesy of kK. L. Jones). 
Production of HoS: Positive. 
Strongly 
Produces streptomycin. 
Source: Soil from Bikini Atoll. 


Type culture: IMRU 3514 


Antagonistic properties: antago- 


nistic. 


30. Streptomyces blastmyceticus Watanabe 
et al., 1957 (Watanabe, K., Tanaka, T. 
Fukuhara, K., Miyairi, N., Yonehara, H.., 
and Umezawa, H. J. Antibiotics (Japan) 
1OA: 39-45, 1957). 


SPECIES OF 


Ky 


erown on casein digest-beef extract 


STREPTOMYCES LS] 


aa” 


es 


lee 


/ 


agar for 12 days, X 3,000-4,000 (Cou1 


Morphology: Sporophores straight. Spores 
oval to spherical, 1 by 1.5 u. 


Sucrose nitrate agar: Growth weak, color- 


less or white. Aerial mycelium poor, white. 


No soluble pigment. 


Glucose-asparagine agar: Growth good, 


= 


colorless, later cream-colored. No aerial my- 


celium. No soluble pigment. 


Calcium citrate-glycerol agar: Growth 


colorless or white, later deep olive-buft. 


182 


Aerial mycelium thin, powdery, white or 
pale yellow to pale olive-buff. 

Nutrient agar: Growth white, later cream- 
colored to light brown. Aerial mycelium 
poor, white to gray. Soluble pigment brown. 

Milk: Growth in the form of ring on sur- 
face, cream-colored to brown. Aerial myce- 
lium white. Soluble pigment brown. Rapid 
peptonization. 

Potato plug: Growth gray to olive-gray. 
Aerial mycelium white. Usually no soluble 
pigment. 

Nitrate reduction: Negative. 

Starch: Hydrolyzed. 

Carbon utilization: Utilizes glucose, fruc- 
tose, galactose, or starch. Grows poorly on 
sucrose, lactose, maltose, or inositol. Does 
not utilize xylose, arabinose, raffinose, rham- 
nose, mannitol, sorbitol, dulcitol, or salicin. 

Antagonistic properties: Produces an 
antifungal agent designated as blastmycin. 

Remarks: Related to S. flavochromogenes. 


31. Streptomyces bobiliae (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 
(Waksman, 8S. A. and Curtis, R. E. Soil Sci. 
1: 121, 1916; 8: 100, 1919). 

Morphology: Elongated sporophores form 
a few close spirals of a dextrorse type. No 
spirals according to Jensen (1930). Spores 
oval and spherical. 

Sucrose nitrate agar: Growth abundant, 
wrinkled, coral-red becoming deep_ red. 
Aerial mycelium scant, white; later absent. 
No soluble pigment. 

Glycerol malate agar: Growth cinnamon- 
buff. No aerial mycelium. 

Glucose-asparagine agar: Growth coral- 
red. No aerial mycelium. 

Nutrient agar: Growth gray, becoming 
brownish to coral-red. No aerial mycelium. 
Soluble brown pigment in presence of glyc- 
erol (Jensen). 

Potato: Growth thin, dry, and wrinkled, 
yellowish, becoming coral-red. No aerial my- 
celium. Soluble pigment grayish to black. 

Gelatin: Growth cream-colored to orange. 


THE ACTINOMYCETEHS, Vol. II 


Aerial mycelium in the form of occasional 
patches of white. Rapid liquefaction. Soluble 
pigment brown. Melanin-positive. 

Milk: Dark brown ring. Peptonization 
without coagulation. 

Starch media: Growth wrinkled, coral-red 
with hyaline margin. Aerial mycelium white. 
Hydrolysis medium. 

Nitrate: Good reduction to nitrite. 

Cellulose: No growth in solution. Good 
growth on plate. 

Invertase: Positive. 

Production of H.S: Positive. 

Temperature: Optimum 37°C. 

Antagonistic properties: Produces 
mented antibiotic cinerubin. 

Habitat: Common in soil. 

Remarks: S. purpurascens is considered by 
Corbaz et al. (1957) as a synonym of S. 
bobiliae, except that the latter no longer 
produces any aerial mycelium or spores. 
IKxrassilnikov (1949) considered this species 


pig- 


as a variety of A. ruber. 
Type culture: IMRU 3310. 


32. Streptomyces bottropensis — IKonink. 
Nederl. Gist et Spirit. (IXonink. Nederland. 
Gist et Spirit. Brit. Pat. 762,736, Dec._5, 
1956). 

Morphology: Aerial mycelium ramified, 
with short, open spirals. Spores cylindrical, 
elliptical to almost spherical, | to 4 by 0.6 
LowlZau: 

Sucrose nitrate agar: Growth abundant, 
reddish. Aerial mycelium limited. Soluble 
pigment brown. 

Glucose-asparagine agar: Growth good, 
yellow. Aerial mycelium limited, white to 
pale gray. No soluble pigment. 

Calcium malate agar: Growth good, vel- 
lowish-brown. Aerial mycelium white-gray. 
No soluble pigment. 

Starch agar: Growth at first pink, later 
darker (pH sensitive; acid-pink, alkaline 
blue). Aerial mycelium limited, white to 
gray. Starch hydrolyzed. 

Glucose 


nutrient agar: Growth folded, 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


yellowish. Aerial mycelium abundant, white. 
No soluble pigment. 

Glucose-yeast extract-peptone — agar: 
Growth yellowish to buff. Aerial mycelium 
white to gray. No soluble pigment. 

Potato agar: Growth smooth, yellowish- 
brown. Aerial mycelium white to  bluish- 
eray. Soluble pigment at first absent, later 
dark. 

Gelatin: Growth on surface good. Ae. ial 
mycelium white. Rapid liquefaction. Soluble 
pigment dark brown. 

Potato: Growth folded, brown to black. 
No aerial mycelium. 

Milk: Growth moderate. No coagulation; 
no peptonization. 

Antagonistic properties: 
biotic B-mycin, active against cocci, gram- 
positive bacteria, and mycobacteria. 

Habitat: Soil. 

Remarks: This is one of the organisms 
that can be either melanin-negative (nutri- 
ent agar, yeast extract agar) or melanin- 
positive (gelatin, potato agar). 


Produces anti- 


33. Streptomyces brasiliensis (Spencer, 
1921) Waksman (Spencer, E. R. Botan. 
Gaz. 72: 285-287, 1921). 

Morphology: Aerial mycelium forms 


straight, branched sporophores. Spores borne 
in chains on free ends of hyphae, oblong, 
1.6. by 0.8 yu. 

Sucrose nitrate agar: Growth at first 
white; after 10 days pale pinkish-buff. Aerial 
mycelium white and dense. No soluble pig- 
ment. 

Glucose-asparagine agar: Growth luxuri- 
ant, color same as on sucrose nitrate agar, 
thallus conspicuously zonated. Aerial myce- 
lium powdery, white to pale pinkish-buff. No 
soluble pigment. 

Glycerol malate agar: Growth spreading 
and not zonated, bordered by submerged 
mycelial bands of varying width, pearl- 
white. Aerial mycelium short, loose, and 
pearl-white. 

Potato: Growth vigorous, crumpled, pale 


183 


pinkish-buff. Aerial mycelium abundant, at 
first white, later pale pinkish-buff. No solu- 
ble pigment. Melanin-negative. 

Nut plugs: Growth vigorous, pale pinkish- 
buff. Aerial mycelium powdery, white. 
Medium not completely but 
much shrunken and blackened. 

Gelatin: Rapid liquefaction. No soluble 
pigment. 

Milk: Rapid coagulation and peptoniza- 


destroyed, 


tion. 
Habitat: 

nuts. 
Remarks: This Streptomyces species is to 

be distinguished from Nocardia brasiliensis, 


Parasitic on kernels of Brazil 


a pathogenic organism. 


34. Streptomyces cacaoi (Waksman, 1932) 
Waksman and Henrici, 1948 (Waksman, S. 
A. In Bunting, R. H. Ann. Appl. Biol. 19: 
515-517, 1932). 

Morphology: Sporophores long; spirals 
long and open, not compact. 

Sucrose nitrate agar: Growth thin, yel- 
lowish, later turning reddish-brown. Aerial 
mycelium light gray to mouse-gray, with 
white edge. No soluble pigment. 


Nutrient agar: Growth brown, covered 
with tiny patches of ivory-colored aerial 
mycelium. 


Potato: Growth abundant, brownish. 
Aerial mycelium white to mouse-gray. Mel- 
anin-negative. 

Gelatin: Growth No aerial 
mycelium. Liquefaction rapid. No soluble 


flocculent. 


pigment. 

Nitrate reduction: Limited. 

Production of HS: Negative. 

Antagonistic properties: Certain strains 
produce an antibiotic designated as cacao- 
mycetin. 

Source: Three strains were isolated from 
cacao beans in Nigeria. They showed slight 
differences, the foregoing description being 
based on one strain. 

Remarks: 
strong diastatic action, no sugar or dextrin 


Strong proteolytic enzymes, 


184 


being left in 1 per cent starch solution after 
a few days. 
Type culture: IMRU 3082. 


35. Streptomyces caelestis DeBoer et al., 
1959 (DeBoer, C., Dietz, A., and Hoeksema, 
H. Canad. Pat. 572,779, March 24, 1959). 

Morphology: Sporophores loosely coiled. 
Spores spherical to oval. 

Sucrose nitrate agar: Growth good. Aerial 
mycelium gray-white. Soluble pigment yel- 
low. 

Nutrient agar: Growth fair to good. Aerial 
mycelium slight pink-white. Soluble pigment 
brown-tan. 

Casein digest-beef agar: 
Aerial mycelium pale glaucous blue. Soluble 
pigment brown-tan. 

Starch agar: No growth. 


Growth good. 


Tyrosine agar: No growth. No aerial my- 
celium. No soluble pigment. 

Potato: Growth good. Aerial mycelium 
grayish to blue-white. Soluble pigment 
brown. 

Gelatin: Growth good. Aerial mycelium 
blue-gray. Soluble pigment brown. Medium 
liquefaction. 

Milk: Growth fair. No soluble pigment. 
No peptonization. 

Nitrate reduction: Negative. 

Production of H.oS: Positive. 

Carbon utilization: Utilizes a variety of 
sugars, dl-inositol, acetate; limited utiliza- 
tion of starch, glycerol, citrate, and succi- 
nate; utilize duleitol, mannitol, 
inulin, sorbitol, and various other organic 


does not 
acids. 

Antagonistic properties: Produces antibi- 
otic celesticetin. 

Habitat: Soil in Utah. 

Remarks: Similar to S. glaucus and SN. 
chartreusis. 


36. Streptomyces caeruleus — (Baldace1) 
Waksman (Baldacci, E. Atti ist. 
univ. Pavia 3: 180-184, 1944). 

Morphology: Sporophores long, straight, 


botan. 


THE ACTINOMYCETES, Vol. II 


branched, not forming any spirals. Spores 
eylindrical 1.0 to. 1.4 by 2.0 toi2-) nu: 

Agar media: Substrate growth colorless. 
Aerial mycelium pigmented, at first white, 
later becoming blue, and finally dark. Solu- 
ble pigment grayish-green. 

Glycerol agar: Grows slowly; light blue in 
color. 

Carrot agar: Growth at first white; later 
becoming blue. Aerial mycelium blue, be- 
coming gradually deep blue, and_ finally 
dark blue. 

Oatmeal agar: Color of growth at first 
white and aerial mycelium blue, gradually 
becoming darker in color. The agar is pig- 
mented grayish-green. 

Gelatin: Growth erayish-blue. 
Either no liquefaction or only slow liquefac- 


slow, 


tion. 
Milk: Weak growth. 
Starch media: Weak greenish growth. 


Bluish-green pigmentation. 


Temperature: Range between 18 and 
30°C. Optimum 24°C. 

teaction: Optimum pH 8 to 10. 

Cellulose: Not utilized. 

Antagonistic properties: Produces anti- 


biotic caerulomycin. 

Habitat: Corn straw and decomposing 
rice straw. 

femarks: Related to S. violaceoruber, S. 
violaceus, and S. viridis. According to Taber 
(1959) the distinctive characteristics of a 
culture that he isolated and identified as 
S. caeruleus are: production of a blue to red 
indicator pigment; requirement of a neutral 
or alkaline reaction for growth and produc- 
tion of oblong to cylindrical spores in straight 
and flexuous chains. It was not chromogenic 
on peptone media but produced HS on 
iron-peptone agar. It did not grow on un- 
buffered potato or carrot plugs, litmus 
milk, and certain synthetic agar media. The 
culture readily utilized glucose, fructose, 
galactose, mannitol, sucrose, xylose, starch, 
and maltose, but did not utilize, or utilized 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


to a limited extent, mannose, ?7-Inositol, 
adonitol, lactose, ribose, raffinose, and cel- 
lulose. 


Type culture: IMRU 3798. 
37. Streptomyces caespitosus Sugawara and 
Hata, 1956 (Sugawara, R. and Hata, T. 
J. Antibiotics (Japan) 9A: 147-151, 1956). 
Morphology: Primary verticils produced. 
Spores oval, 1.3 to 0.5 by 0.3 to 0.5 u. 
Sucrose nitrate agar: Growth hyaline, 
colorless to faint yellowish-brown. Aerial 
mycelium white to yellowish-gray to green- 
ish-yellow. Soluble pigment faint yellow. 
Calcium malate Growth 
with yellow-brownish center, changing to 
dark greenish-yellow to dull yellow. Aerial 
yellowish 


agar: colorless 


mycelium white, with tinge, 
greenish-yellow at the margin. Soluble pig- 
ment faint yellow, pinkish shade in some 
cultures. 

Glucose-peptone agar: humid, 
wrinkled, cracked in the center; colorless, 
becoming greenish-yellow-gray, — reddish- 
gray, to dark gray. Aerial mycelium thin, 
gray. Soluble pigment reddish-brown. 

Starch agar: Growth scanty, colorless to 


Growth 


faint yellowish-brown, or yellow to orange- 
yellow. Aerial mycelium cottony, white, 
cream with lavender patch in the center, 
becoming yellowish-gray. Soluble pigment 
faint yellowish-brown. 

Nutrient agar: Growth yellow-brown to 
gray to dark gray. Aerial mycelium gray. 

Potato: Growth cream to brownish, center 
light greenish-yellow. Aerial mycelium white 
to grayish or gray with pale olive tinge. 
Soluble pigment absent, or dark brown, or 
grayish-brown. 

Milk: Surface ring yellow to pale yel- 
lowish-brown. No aerial mycelium. Soluble 
pigment pale brown. 

Gelatin: Growth cream-colored turning 
greenish-yellow to reddish-yellow. Aerial 
mycelium white to yellow. Soluble pigment 
yellowish-brown. Rapid liquefaction. 
Tyrosinase reaction: None. 


LS5 


Nitrate reduction: Positive. 

Starch: Hydrolysis. 

Carbon utilization: Utilizes various car- 
bohydrates; does not utilize xylose, rham- 
nose, raffinose, arabinose, mannitol, salicin, 
dulcitol, inulin, acetate, and succinate. 


Antagonistic properties: Produces anti- 
biotic mitomycin, active upon certain 
neoplasms. 


temarks: Closely related to S. kitasatoen- 
sis and S. hachijoensis. 


38. Streptomyces caiusiae Dhala et al., 
1957 (Dhala, 8S. A., Poonawalla, F. M., and 
Bhatnagar, 8S. S. J. Sci. & Ind. Res. 16C: 
76-80, 1957). 

Morphology: Aerial hyphae short and 
straight; frequently clusters are produced, 
subdividing at the distal portions into chains 
of spores. No spirals formed either in syn- 
thetic or nonsynthetic media; often tips of 
the aerial hyphae slightly curved. Spores 
round to oval, 0.6 to 1.4 by 0.4 to 0.8 yu. 
nitrate agar: Colonies round, 
convex, tough, with smooth surface when 
unsporulated; citron-yellow, later turning 
brown. Aerial mycelium white, turning yel- 
lowish, then gray. Soluble pigment at first 
yellow but later darkened to a brown tinge. 

Glucose-asparagine agar: Growth citron- 
vellow. Aerial mycelium white. Soluble pig- 


Sucrose 


ment yellowish-brown. 
Nutrient agar: Growth 
irregular margins and radial ridges in old 
cultures. White aerial mycelium. 
Starch agar: 
after 2 days; on further incubation, they 


wrinkled, with 


Colonies smooth, colorless 


become large and wrinkled with radiating 
ridges. Aerial mycelium white, turning gray. 
Soluble pigment brown. Starch weakly hy- 
drolyzed. 

Potato: Growth luxuriant, citron-yellow. 
Aerial mycelium white, turning gray. Plug 
turns black. 

Milk: Pellicle produced. Coloration of 
milk brownish to black. Peptonization posi- 
tive. 


186 


Gelatin: Sediment buff-colored. Liquefac- 
tion medium. 

Tyrosinase reaction: Positive. 

Nitrate reduction: None. 

Carbon sources: Sugars readily utilized, 
with the exception of acetate, benzoate, 
cellulose, dulcitol, 2-inositol, and salicylate. 

Antagonistic properties: Active primarily 
upon gram-negative bacteria and fungi. 

Remarks: Closely related to S. antzbi- 
oticus. 

39. Streptomyces californicus (Waksman 
and Curtis, 1916) Waksman and Henrici 
(Waksman, S. A. and Curtis, R. E. Soil Sci. 


1: 22, 1916;\Waksman, 8. A. zbed. 8: 104, 
1919). 
Synonyms: 

Streptomyces puniceus Finlay and Sobin, 
1950. 
Streptomyces vinaceus Mayer et al., 
1951. 
Streptomyces floridae Bartz et al., 1951. 
Streptomyces griseus var. purpureus 
Burkholder e¢ al., 1955. 
Streptomyces purpureus (Burkholder, 


1955) Waksman, 1959. 

Morphology: The original culture was re- 
ported to form sporophores with long, nar- 
row, open sinistrorse spirals. According to 
Okami, however, the 
straight. Recent examinations of the original 
culture of Waksman and Curtis (Burkholder 
et al., 1955) did not reveal any spirals either. 

Sucrose nitrate agar: Growth spreading, 
Aerial mycelium 
powdery, light neutral gray to ash-gray. No 
soluble pigment. 


sporophores — are 


vinaceous-colored. 


Glucose-asparagine agar: Growth — re- 
stricted, much folded, cream-colored, with 
sulfur-yellow tinge. 

Nutrient 


yellowish to cream-colored, Melanin-nega- 


agar: Growth thin, restricted, 
tive. 

Starch Growth 
center with colorless to gray margin. Hy- 


spreading, pink 


agar: 


drolysis rapid. 


THE ACTINOMYCETES, Vol. II 


Potato: Growth glossy, yellow to red, 
turning red-brown. 

Gelatin: Growth gray, moist, abundant. 
No soluble pigment. Liquefaction medium. 

Milk: Surface growth faint, brownish. 
Coagulation and slow peptonization. 

Nitrate reduction: Positive. 

Production of H.S: Negative. 

Cellulose: Growth scant but definite. 

Carbon utilization: According to Burk- 
holder, the various strains utilize p-xylose, 
b-glucose, p-galactose, p-fructose, cellobiose, 
p-maltose, p-mannitol, and starch. Growth 
poor with L-arabinose, L-rhamnose, p-lac- 
tose, sucrose, D-raffinose, dulcitol, 2-inositol, 
and salicin. 

Temperature: Optimum 37°C. 

Invertase: Positive. 

Antagonistic properties: Routien and 
Hofmann (1951) first demonstrated that 
cultures of S. californicus are capable of 
producing viomycin. The same antibiotic 
was found to be produced by other strains 
of this organism. 

Habitat: Soil. 

temarks: Waksman and Curtis reported 
the production of spirals in their original 
description of S. californicus (see also Waks- 
man, 1919). Several authors who studied 
the S. californicus culture more recently de- 
scribed the aerial mycelium as straight to 
wavy to strongly flexuous (Burkholder et al., 
1955; Kutzner, 1956; Ettlinger et al., 1958). 
Since the other properties of the now-avail- 
able S. californicus fit with the original de- 
scription, it might be assumed that the 
strongly flexuous aerial hyphae were con- 
sidered as spirals originally. In 1955 Burk- 
holder et al. made a comparative study of 
several viomycin-producing organisms which 
were originally described under the names 
S. floridae, S. puniceus, S. vinaceus, S. calr- 


fornicus, and several others. All these cul- 


tures behaved in a similar manner, with 
only minor differences between this group 
and S. californicus ATCC 3312; namely, 


DESCRIPTION OF SPECIES OF STREPTOMYCES 187 


“with the exception of ATCC 3312, all 
isolates liquefy gelatin rapidly and produce 
viomycin or similar antibiotic compounds.” 
No studies seem to have been made, how- 
ever, of antibiotics produced by S. californi- 
cus ATCC 3312. Only because the original 
description of S. californicus gave spiral for- 
mation and these organisms did not, the 
several viomycin-producing or 
the S. californicus ATCC 33 
scribed as a variety of S. griseus, namely 
S. griseus var. purpureus. This was due to 
the fact that S. griseus had the same mor- 


ganisms and 
12 were de- 


phology and color of the aerial mycelium, 
and because several streptomycin-producing 
strains are known to form also a red-gray 
color in the substrate growth. 

Type culture: IMRU (ATCC) 3312. 

40. Streptomyces calvus Backus et al., 
1957 (Backus, E. J., Tresner, H. D., and 
Campbell, T. H. Antibiotics & Chemother- 
apy 7: 532-541, 1957). 

Morphology: Sporophores form 
loose spirals. Spores globose to elongated, 
0.6 to 1.0 by 1.0 to 1.8 » (Fig. 34). 

Sucrose nitrate agar: Growth cream-col- 


short 


ored to yellowish. Aerial mycelium scanty, 
white to gray. 

Glucose-asparagine agar: Growth ivory- 
yellow. Aerial mycelium scanty, white. 

Calcium malate agar: Growth colorless to 
vellow. Aerial mycelium scanty white to 
gray. Crystalline pellets formed in growth 
ZONES. 

Starch agar: Growth colorless to yellowish. 
Aerial mycelium white to mouse-gray. 

Nutrient agar: Substrate growth light yvel- 
low. Aerial mycelium scanty, white. No 
soluble pigment. Melanin-negative. 

Potato plug: Growth gray. Aerial myce- 
hum scanty, white to light gray. Plug dis- 
colored. 

Gelatin: Growth colorless to yellow. No 
aerial mycelium. Partial liquefaction. No 
soluble pigment. 


Milk: Growth colorless to yellow. Coagu- 
lation and moderate peptonization. 

Cellulose: Growth yellow. No decomposi- 
tion of cellulose. 

Production of HoS: Negative. 

Carbon utilization: d-fructose, 7-inositol, 
lactose, d-mannitol, d-raffinose, /-rhamnose, 
sucrose, d-trehalose, and d(+-)-xylose readily 
utilized; /-arabinose, d-melibiose, and_ sali- 
cin utilized poorly; dextrin, esculin, deme- 
lezitose, and adonitol not utilized at all. 

Antagonistic properties: Produces 
nucleocidin, an antibiotic possessing anti- 
trypanosomal properties. 

Habitat: Soil in India. 

Remarks: This organism is closely related 
to S. annulatus. 


41. Streptomyces candidus (Krassilnikov, 
1941) Waksman (Not Streptothrix candida 
Petruschky). CUsrassilnikov, N. A. Actino- 
mycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 49, 1941). 

Morphology: Sporophores long, straight 
or wavy, but never forming spirals; occa- 
sionally arranged in broom-shaped_ bodies 
or fascicles. Spores oblong to cylindrical 
(‘fragmentation spores”), 1.0 to 2.0 by 
0.6 to 0.8 x. 

Sucrose nitrate 
Aerial mycelium velvety, white. No soluble 


agar: Growth colorless. 
pigment. 

Nutrient agar: Growth good, lichenoid 
or smooth. Aerial mycelium whitish. Mela- 
nin-negative. 

Gelatin: Slow liquefaction. Melanin-nega- 
tive. 

Potato: Growth colorless, lichenoid. Aerial 
mycelium poorly developed. No soluble pig- 
ment or brownish. 

Milk: No coagulation; good peptoniza- 
tion. 

Starch: Rapid hydrolysis. 

Cellulose: Good growth. 

Nitrate reduction: Positive. 

Sucrose: Inversion. 

Production of HS: Negative. 


188 


THE ACTINOMYCETES, Vol. II 


FriGcure 34. Sporophores of S. caluus (Reproduced from: Backus, E. J. et al. Antibiotics & Chemo- 


therapy 7: 536, 1957). 


Antagonistic properties: Weak. 

Habitat: Soil. 

Remarks: Certain varieties of this species 
have also been described. This is true, for 
example, of A. fasciculus, which Krassilni- 
kov himself considered as a variety of S. 
candidus; it 1s true of A. farinosus 
Krassilnikov and of A. candidus var. albo- 
roseus described by Gause et al. (1957). 
S. nitrosporeus Okami (1952) appears to be 
closely related, if not identical to it. Ett- 
linger et al. (1958) consider this organism 


also 


as related to S. griseus. 

Type culture: IMRU 3416. 

42. Streptomyces canescus Hickey et al., 
1952, (Hickey, “Ri J.;- Corum, ©. J.) "midy, 


P, H., Cohen; I.R:; Nagers U.F= Band 


Kropp, E. Antibiotics & Chemotherapy 
2: 472-483, 1952). 


Morphology: Sporophores straight — or 
curved, not forming any spirals. Spores 
globose, 1.0 to 1.3 by 1.3 to 2.6 p (ig. 35) 

Calcium malate agar: Growth gray to 
rose-gray; reverse yellow to tan. No soluble 
pigment. 

Yeast extract-casein digest agar: Growth 
effuse to convex, edge filamentous; reverse 
brown. Aerial mycelium powdery, varying 
from gray-white to gray. No soluble pig- 
ment. 

Acid-glucose-peptone Growth at 
first white, then tan. Aerial mycelium faintly 


agar: 


z 


DESCRIPTION OF SPECIES OF STREPTOMYCES LS9 


greenish, produced after 14 days. Amber Milk: Soft, rennet curd formed at 36°C 
pigment diffused throughout medium. after 48 hours; completely peptonized in 12 
Kee medium: Growth tan, wrinkled. No days. 
sporulation after 10 days; limited white Starch: Hydrolysis strong. 
sporulation observed in 14 days. Soluble Nitrate reduction: Negative. 
pigment brown. Very slow liquefaction after Production of H.S: Negative. 
28 days. Temperature: Optimum 36°C 
Potato: Growth lhght gray, wrinkled. Carbon utilization: Utilizes glucose, arabi- 
Soluble pigment deep brown. nose, trehalose, xylose, sucrose, maltose, 
Gelatin: Liquefaction rapid. Soluble pig- galactose, dextrin, soluble starch, mannitol, 
ment deep brown. glycerol, and salicin. No growth with sor- 


¥ 


sl], A : 


Fiat RE 35. S. canesc us, grown on casein digest beef extract agar for 12 days Xx 3.000—4.000 (Courtesy 


Le 


@ 


of kK. L. Jones 


190 


bose, melezitose, dulcitol, rhamnose, sorbi- 
tol, melibiose, phenol, raffinose, and lactose. 
Antagonistic properties: Produces  anti- 
fungal antibiotic ascosin. 
Source: Contaminated fungus plate. 
Remarks: This species is now included 
with S. coelicolor (Kutzner and Waksman, 
1959). 
Type culture: IMRU 3782; NRRL 2419. 


43. Streptomyces canus Heinemann et al., 
1953 (Heinemann, B., Kaplan, M. A., Muir, 
R: D:..and> Hooper. i... Antibiotics: .& 
Chemotherapy 3: 1239-1242, 1953). 

Morphology: Aerial mycelium forms nu- 
merous loosely wound spirals. Spores spher- 
oidale 0 toa..2 by 1:6 towl.8: a: 

Sucrose nitrate agar: Growth moderate, 
wrinkled, yellow-brown. Aerial mycelium 
secant. No soluble pigment. 

Glycerol-asparagine agar: Growth abun- 
dant, cream-colored, turning russet-brown 
with aging. Aerial mycelium abundant, 
slate-gray. Soluble pigment amber. 

Calcium malate agar: Growth moderate, 
golden colored. Aerial mycelium scant. No 
soluble pigment. 

Nutrient agar: Growth abundant, yellow. 
Aerial mycelium white to light yellow. Solu- 
ble pigment faint yellow. 

Potato: Growth abundant, cream-colored. 
No aerial mycelium. Slight reddish-brown 
darkening of the potato. Melanin-negative. 

Gelatin: Moderate liquefaction at 26°C 
in 14 days. No soluble pigment. 

Milk: Alkaline with no coagulation; slight 
peptonization in 14 days. 

Starch: Hydrolysis in 96 hours at 30°C. 

Nitrate reduction: Positive in 96 hours 
at 30°C. 

Carbon utilization: Good growth with 
arabinose, rhamnose, xylose, dextrose, galac- 
tose, fructose, cellobiose, lactose, maltose, 


sucrose, dextrin, inulin, raffinose, soluble 
starch, glycerol, inositol, mannitol, and 


sodium salicylate. No growth observed with 
dulcitol, sorbitol, sodium acetate, sodium 


THE ACTINOMYCETES, Vol. II 


citrate, sodium formate, sodium malate, 
sodium oxalate, sodium tartrate, or sodium 
succinate. 

Antagonistic properties: Produces the 
antibiotic amphomycin, active against gram- 
positive bacteria. 

Habitat: Soil. 

Type culture: ATCC 12,237. 

44. Streptomyces carnosus (Millard and 
Burr, 1926) Waksman and Henrici (Millard, 
W. A. and Burr, S. Ann. Appl. Biol. 13: 
580, 1926). 

Morphology: Spores cylindrical, 1.0 by 
OFT amis. 

Sucrose nitrate agar: Growth pale smoky- 
gray to olive-gray. Aerial mycelium abun- 
dant, gray. Colorless guttation drops appear 
over the whole surface. Soluble pigment 
ivory-yellow to cartridge-buff. 

Potato: Growth lichenoid. Aerial 
celium gray to brownish with white spots. 
Plug becomes colored gray to black. 

Gelatin: Surface growth. Aerial mycelium 
white in center, gray at margin. Rapid lique- 
faction. Soluble brown pigment. 

Milk: Surface growth good. No aerial my- 
celium. Positive coagulation and peptoniza- 
tion. 

Starch: Hydrolysis. 

Tyrosinase reaction: Negative. 

Nitrate reduction: Positive. 

Habitat: Potato scab. 


my- 


45. Streptomyces catenulae Davisson and 
Finlay, 1959 (Davisson, J. W. and Finlay, 
A. C. U.S. Pat. 2,895,876, July 21, 1959). 

Morphology: Growth with 
smooth edge. Sporophores in form of short 


wrinkled 


clusters; a few tight spirals. Spores oval to 
cylindrical, 1.0 by 1.3 wu. 

Agar media: Growth dark brown to dark 
greenish-brown. Aerial mycelium dark olive- 
gray to brown. 

Sucrose nitrate agar: Growth transparent, 
white. Aerial mycelium light gray. No solu- 


ble pigment. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 19] 


Calcium malate agar: Growth poor. Aerial 
mycelium mouse-gray, with some white. Cal- 
cium malate digested. 

Nutrient agar: Growth pale yellow. Aerial 
mycelium white, turning pale gray. Soluble 
pigment pale yellow. Nonchromogenic. 

Glucose-yeast extract-beef-peptone agar: 
Growth dark brown. Aerial mycelium olive- 
gray with white. Soluble pigment medium 
brown. 

Starch agar: Growth 
Aerial mycelium poor, mouse-gray. Good 
hydrolysis. 

Gelatin: Growth moderate. Aerial myce- 
lium buff, some white. No soluble pigment. 
Poor liquefaction. 

Potato: Growth good, greenish. Aerial my- 
celium pale olive to smoke-gray to brown. 
Soluble pigment dark greenish or absent. 

Nitrate reduction: None. 

Antagonistic properties: Produces 
biotic catenulin. 

Habitat: Soil. 

Type culture: ATCC 12,476. 

46. Streptomyces cavourensis Giolitti, 1958 
(Giolitti, G. Belgian Pat. 560,930, March 18, 
1958*). 

Morphology: Aerial mycelium produces 
spirals on certain media. Spores spherical to 
elliptical. 

Sucrose nitrate agar: Growth yellowish. 
Aerial mycelium chalky white to yellowish. 


brownish-orange. 


anti- 


No soluble pigment. 

Glycerol-asparagine agar: Growth hazel- 
colored. Aerial mycelium whitish. Soluble 
pigment faint brown. 

Calctum malate agar: Growth scanty, 
yellow-brownish. Aerial mycelium scanty, 
white. Soluble pigment scarce, yellow-brown- 
ish. 

Nutrient 


Aerial mycelium scanty, chalky white to yel- 


agar: Growth orange-brown. 
lowish. Soluble pigment light brown. 


Glucose agar: Growth brown, wrinkled. 


* Supplemented by personal communication. 


Aerial mycelium white-yellowish. Soluble 
pigment dark brown. 

Potato agar: Growth dark brown. Aerial 
mycelium gray with dark yellow dots. Solu- 
ble pigment brown. 
light brown, 
wrinkled. Aerial mycelium gray with brown 
patches. Soluble pigment light brown. 

Starch agar: 
brownish. Aerial mycelium scanty, white- 
grayish. Soluble pigment brownish. Strong 
hydrolysis. 

Gelatin: Growth scanty, brown, wrinkled. 
Aerial mycelium scanty, gray. Soluble pig- 


Oatmeal Growth 


agar: 


Growth scanty, yellow- 


ment brownish. Liquefaction fairly good. 

Potato: Growth brown with yellow edges, 
wrinkled. Aerial mycelium grayish. Soluble 
pigment brown. 

Milk: Growth consists of white to yellow 
ring around surface. Positive coagulation and 
peptonization. 

Nitrate reduction: Negative. 

Antagonistic properties: Produces flaven- 


somycin, an antibiotic active against fila- 
mentous and yeast-like fungi, and to a 


certain extent some gram-positive bacteria. 
Very active against some insects. 

Type culture: IMRU 3758. 

47. Streptomyces celluloflavus Nishimura 
et al., 1953 (Nishimura, H., Kimura, T., and 
Kuroya, M. J. Antibiotics (Japan) 6A: 57— 
65, 1953). 

Morphology: Sporophores straight with a 
few flexible, hooked spirals. Spores nearly 
spherical, 1.0 by 0.9 x. 

Sucrose nitrate agar: Growth glossy, de- 
veloping deep into medium, later becoming 
yellow. Soluble pigment faint sulfur-yellow. 


malate agar: Growth yellow, 


Glycerol 
later turning white to pale olive-buff with 
blackish center. Aerial mycelium cottony, 
white, with grayish patches, later turning 
olive-buff. Soluble pigment yellow. 

Glucose-asparagine agar: Growth cream 
to yellow. Aerial mycelium scant, cottony, 


192 THE ACTINOMYCETES, Vol. II 


white to gray. Soluble pigment sulfur-yel- 
low. 

Nutrient agar: Growth olive-buff, turn- 
ing colorless. Aerial mycehum scant, cot- 
tony, white to grayish. Soluble pigment 
yellow with tinge of green to gold. 

Potato: Growth wrinkled, deep olive-buff. 
Aerial mycelium white to olive-buff. Soluble 
pigment deep olive-buff. 

Gelatin: Growth ivory-yellow to olive-buff 
on surface of liquefied layer. No aerial my- 
celium. Faint brownish pigment. Rapid to 
medium liquefaction. 

Milk: Growth yellow to dark olive-buff. 
Aerial mycelium white. Soluble pigment red- 
dish-brown. Coagulation and rapid peptoni- 
zation. 

Tyrosine medium: Growth ivory-yellow to 
cream-buff. Aerial mycelium absent or scant 
white. Soluble pigment greenish-yellow. 

Cellulose agar: Growth poor. Soluble pig- 
ment yellow. 

Production of HS: Negative. 

Antagonistic properties: Produces  thio- 
lutin, aureothricin. 

Habitat: Soil. 

48. Streptomyces cellulosae — (IXrainsky, 
1914) Waksman and Henrici, 1948 (x<rain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
IT., 41: 683-688, 1914). 

Description after Jensen, H. L. Soil Sei. 
30: 65, 1930. 

Morphology: 
spiral formation. Spores almost spherical, 1.5 


Sporophores straight; no 


uw in diameter. 

Sucrose nitrate agar: Growth at first trans- 
parent, becoming lemon-yellow. Aerial my- 
celium light gray, later deep slate-gray. Sol- 
uble pigment may be lemon-yellow. 

Calcium malate agar: Colonies yellowish; 
aerial mycelium gray to white-gray. Soluble 
pigment yellow. 

Glucose-asparagine agar: Growth abun- 
dant; aerial mycelium gray. Soluble yellow 
pigment, especially with high nitrogen con- 
centration. 


nutrient Good substrate 
growth, at first cream-colored, later sulfur- 


vellow. Aerial mycelium white, later gray. 


Glucose agar: 


Potato: Growth light cream-colored, later 
often yellow. Aerial mycelium white, later 
slate-gray. 

Gelatin: Growth yellowish-gray to gray- 
ish-black. Rapid liquefaction. 

Milk: Rapid coagulation and peptoniza- 
tion. 

Cellulose: Growth good. 

Esculin: Hydrolysis. 

Starch: Diastatic action strong. 

Nitrate: Reduction weak. 

Invertase: Negative. 

Production of HS: Negative. 

Temperature: Optimum 30-35°C. 

Pigment: Soluble in alcohol and other or- 
ganic solvents. 

Antagonistic properties: Produces anti- 
biotics fungichromin and actinomycin. 

Habitat: Very common in soil. 

Remarks: The culture described by Krain- 
sky (1914) produced an aerial mycelium of a 
eray color (‘like diastaticus’’). Later, how- 
ever, Krainsky’s culture was found to pro- 
duce a grayish-yellow aerial mycelium like 
the streptomycin-producing S. griseus (Ett- 
linger ef al., 1958); these authors also con- 
sider the S. cellulosae strains obtained from 
ATCC and NRRL as closely related to S. 
griseus. Most probably, one of these cultures 
was used in the studies of Tresner and Dang: 
(1958), who mention a yellow-cream-buff 
color of the aerial mycelium. In contrast to 
these cellulosae strains now available for 
comparison, Jensen (1930a) described five 
soil isolates as A. cellulosae with a distine- 
tive slate-gray aerial mycelium, in agree- 
ment with Krainsky’s description. Krassilni- 
kov (1949) considers this organism as a 
variety of A. flavus. 

Type culture: IMRU 3313, 3780. 


49. Streptomyces chartreusis Calhoun and 
Johnson, 1956 (Calhoun, Ik. M. and John- 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


son, L. E. Antibiotics & Chemotherapy 6: 
994-298, 1956). 

Morphology: Aerial hyphae branch pro- 
fusely producing closed or open spirals, 
sinistrorse and dextrorse, depending on com- 
position of medium. Spiral chains consist of 
3 to 7 turns. Spores powdery, blue-gray to 
blue-green, depending on the medium. Most 
spirals occur singly; a few are found in 
groups of two or three measuring 5 to 20 u 
in length and 3 to 5 uw in width. Spores 
spherical to oval, 1.0 to 1.5 uw in diameter. 

Sucrose nitrate agar: Growth profuse, 
raised and somewhat wrinkled, honey-col- 
ored. Aerial mycelium of young colonies 
white to pale gray; older colonies have blue 
center. 

Glucose-asparagine agar: Growth profuse, 
blue-gray. Soluble pigment yellow. 

Starch agar: Growth profuse. Center of 
colonies blue-green, edges white. No soluble 
pigment. Good hydrolysis. 

Nutrient agar: Growth profuse. Center of 
colonies blue-gray, edges powdery white. No 
soluble pigment. 

Potato: Growth light, raised with some 
wrinkling. Center of colonies blue-gray with 
white edges. No soluble pigment. 

Gelatin: Growth blue-green in center with 
white edges. Soluble pigment yellow-green 
to black. Slow liquefaction. Melanin-posi- 
tive. 

Milk: Growth moderate, blue-gray and 
white. Slow peptonization. 

Nitrate broth: Blue-green ring and white 
pellicle. Soluble pigment vellow-tan. Nitrate 
strongly reduced. 

Production of H.S: Positive. 

Antagonistic properties: Produces anti- 
biotic chartreusin. 

Xtemarks: Ettlinger et al. (1958) considered 
this species to be related to S. vzridochromo- 
genes. 


50. Streptomyces chibaensis Suzuki et al., 
1958 (Suzuki, 8., Nakamura, G., Okuma, K.., 


195 


and Tomiyama, Y. 
11LA: 81-83, 1958). 
Morphology: Sporophores wavy, produc- 


J. Antibiotics (Japan) 


ing numerous small spirals. 

Sucrose nitrate agar: Growth slow, pene- 
trating into medium, yellow reverse. At first 
no aerial mycelium; later mycelium pro- 
duced, white becoming gray, then reddish- 
gray or black-buff. Soluble pigment shght 
vellow. 

Nutrient 
cream-colored or yellow. Aerial mycelium 


agar: Growth good. Reverse 
powdery, white. Soluble pigment absent or 
slightly yellow. 

Glucose-asparagine agar: Growth good, 
vellow, later becoming brown. Aerial myce- 
lium powdery, white, later becoming dark 
brown. Soluble pigment yellow. 

Starch agar: Growth good, white, later be- 
coming olive-yellow. Strong hydrolysis of 
starch. 

Potato: Growth good, raised. Aerial my- 
celium white or cream-colored, powdery. No 
change in color of plug. 

Gelatin: Growth on surface and in medium 
poor. No aerial mycelium. Liquefaction ab- 
sent or limited. Melanin-negative. 

Milk: Growth good; white to cream-col- 
ored pellicle. No aerial mycelium. No soluble 
pigment. Peptonization slow. 

Carbon sources: Utilizes readily a variety 
of carbon sources, but not inulin. 

Antagonistic properties: Produces anti- 
biotic cellocidin, active against gram-positive 
and gram-negative bacteria; this antibiotic 
also possesses anticancer properties. 

Habitat: Soil in Japan. 

temarks: The organism is closely related 
to S. flavus. 


51. Streptomyces chrysomallus Lindenbein, 
1952 (Lindenbein, W. Arch. Mikrobiol. 17: 
361-383, 1952). 

Morphology: Substrate growth soft, con- 
sisting of long, branching hyphae, with nu- 


merous staining granules. Sporophores long, 


194 


straight; no spirals. Spores oval to elliptical; 
surface smooth. 

Glycerol nitrate agar: Growth light yellow. 
Aerial mycelium powdery, white. Soluble 
pigment golden yellow. 

Glucose-asparagine agar: Growth smooth, 
colorless to yellowish. Aerial mycelium 
powdery, white. Soluble pigment faint yel- 
low. 

Glycerol malate Growth thin, 
smooth, colorless to light yellow. Aerial my- 
celium powdery, grayish-white. 

Nutrient agar: Growth poor, shiny, golden 
yellow. Aerial mycelium white, powdery. 
Soluble pigment golden yellow. Melanin- 


agar: 


negative. 

Glucose-peptone agar: Growth yellowish 
with tinge of orange. Aerial mycelium gray- 
ish-white. Soluble pigment lhght yellow to 
golden yellow. 

Starch-casein agar: Growth colorless, with 
yellowish reverse. Aerial mycelium powdery, 
chalk-white. Strong hydrolysis of starch. No 
soluble pigment. 

Potato: Growth heavy, yellow, becoming 
brownish-yellow or orange. Aerial mycelium 
cottony white to yellowish-white. 

Gelatin: Surface growth heavy, hght to 
dark yellow. Aerial mycelium white. Soluble 
pigment yellow-brown to deep brown, only 
in liquefied portion. Strong liquefaction. 
Melanin-negative. 

Milk: Growth colorless, with hight yellow 
reverse. Aerial mycelium cottony, snow- 
white, becoming yellowish. Coagulation 
shght. Strong peptonization. 

Cellulose: Growth very weak. 

Production of HoS: Negative. 

Antagonistic properties: Produces actino- 
mycin C; some strains also produce the 
antifungal cycloheximide. 

Habitat: Soil. 

Remarks: Ettlinger et al. (1958) considered 
it as a member of the S. griseus group. A 
complete description of this organism was 
(1958). Frommer 


also given by Frommer 


THE ACTINOMYCETES, Vol. II 


(1959) described a variety of this organism 
under the name fumigatus. It differed from 
the type species by producing a mouse-gray 
aerial mycelium on synthetic media, no 
aerial mycelium on potato and gelatin, and 
by displaying more limited proteolytic prop- 
erties. 
Type culture: IMRU 3657. 


52. Streptomyces cinereoruber Corbaz et al., 
1957 (Corbaz, R., Ettlinger, L., Keller- 
Schierlein, W., and Zihner, H. Arch. Mikro- 
biol. 25: 825-332, 1957). 

Morphology: Sporophores straight; no 
spirals. Spores shghtly elongated, 0.9 to 2 by 
0.7 to 1 w; surface of spores smooth. 

Glycerol nitrate agar: Growth thin, light 
carmine-red, in 7 days dark red. Aerial my- 
celium ash-gray. Soluble pigment lght car- 
mine. 

Glucose-asparagine agar: Substrate 
erowth thin, greenish-gray to bluish-gray. 
Aerial mycelium ash-gray. No soluble pig- 
ment. 

Gelatin: Surface growth hght carmine to 
light brown. Aerial mycelium light gray. Sol- 
uble pigment red-brown. Medium liquefac- 
tion. Melanin-positive. 

Starch agar: Vegetative growth coral-red. 
Aerial mycelium ash-gray. Soluble pigment 
carmine. Hydrolysis limited. 

Potato: Growth lichenoid, brownish-yel- 
low. Aerial mycelium ash-gray. Soluble pig- 
ment bluish-gray. 

Milk: Pellicle ght brown with sparse 
aerial mycelium, powdery, white-gray. Coag- 
ulation and peptonization. Reaction turns 
acid. 

Carbon utilization: Utilizes xylose, arabi- 
nose, and other sugars. Does not utilize L- 
rhamnose, b-fructose, raffinose, mulin, d- 
sorbitol. 

Antagonistic properties: Produces anti- 
biotic rhodomycin. 

Remarks: This organism is closely related 
to S. bobiliae and S. purpurascens. S. cine- 
reoruber var. fructofermentans is a variety, 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


based on differences in sugar utilization, and 
produces the antibiotic cinerubin. 


53. Streptomyces cinnamomeus Benedict 
et al., 1954 (Benedict, R. G., Dvonch, W., 
Shotwells OO) i... eEnidnam: ©:-Gy and Lin- 
denfelser, L. A. Antibiotics & Chemotherapy 
2: 591, 1952; 4: 1140, 1954). 

The correct name of this organism is S. 
cinnamomeus ft. cinnamomeus. 

Morphology: Sporophores straight; later 
descriptions indicate verticil formation. 
Spores globose, 0.6 uw (Fig. 36). 

Sucrose nitrate agar: Growth colorless to 
white to cream-colored. Aerial mycelium 
white to light cinnamon. 

Glucose-asparagine agar: Growth color- 
less; light greenish-yellow to dull yellowish- 
orange in reverse. Aerial mycelium white to 
cinnamon. 

Nutrient agar: Growth cream-colored to 
light lemon-yellow. No aerial mycelium. No 
soluble pigment. 

Oatmeal agar: Growth tough, leathery, 
yellowish-green to cream-yellow. Aerial my- 
celium floccose, pale violet to faint cinna- 
mon. Exudate tan to white. 

Starch agar: Growth colorless to brownish. 
Aerial mycelium white. Hydrolysis. 

Potato: Growth grayish-white to yellow- 
green to light brown. Aerial mycelium light 
gray to gray. No soluble pigment. 

Gelatin: Growth flocculent, dirty yellow to 
white. Aerial mycelium cretaceous. No solu- 
ble pigment. Rapid liquefaction. 

Milk: Ring light brown. Aerial mycelium 
limited, white. Rapid peptonization. 

Carbon utilization: Utilizes xylose, fruc- 
tose, inositol, starch, dextrin, galactose, and 
maltose. Does not utilize arabinose, rham- 
nose, duleitol, and salicin. 

Nitrate reduction: Negative. 

Production of HS: Negative. 

Temperature: Good growth at 25-37°C. 

Antagonistic properties: Produces cinna- 
mycin, a polypeptide antibiotic. 

Source: Japanese soil. 


195 


FriGure 36. Hyphae of S. c¢nnamomeus showing 


character of verticils of branches 
(Reproduced from: Duggar, B. M. ef al. 


N. Y. Acad. Sci. 60: 85, 1954). 


sporogenous 
Ann. 


Remarks: Pridham ef al. (1956) described 
a second form under the name of S. cinna- 
momeus f. azacoluta; it produced a shell-pink 
aerial mycelium on starch agar and an anti- 
biotic, duramycin. 

Type culture: IMRU 3664. 


54. Streptomyces cinnamonensis Okami, 
1953 (Okami, Y., Maeda, K., Kosaka, H.., 
Taya, O., and Umezawa, H. Japan. J. Med. 
Se. Biol. 6: 87-90, 1953). 

Morphology: Sporophores long, flexible, 
hooked, but no true spirals. Spores elliptical 
to oval. 

Nutrient agar: Growth colorless to dark. 
No aerial mycelium. Soluble pigment absent 
or slightly brown. 

Glycerol agar: Growth colorless. Scant 
white aerial mycelium or white with pale 
cinnamon-pinkish to light brownish-vina- 
ceous tinge. No soluble pigment. 

Glucose-asparagine agar: Growth colorless 
to light Aerial 
white to white-pinkish-cinnamon. No soluble 


cream-colored. mycelium 


pigment. 


196 


Potato: Growth dark to light cream-col- 
ored. No aerial mycelium. No soluble pig- 
ment; later, black pigment produced around 
growth. 

Gelatin: Growth colorless to dark brown- 
ish. Aerial mycelium in white 
patches. Soluble pigment brown. No or very 


form of 


slow liquefaction. 

Milk: Growth cream-colored to brownish 
surface ring. Aerial mycelium absent or 
scant white. Soluble pigment 
slightly brown. Coagulation and peptoniza- 
tion absent or very slow. 

Starch agar: Growth colorless. Aerial my- 


absent or 


celium white with pinkish tinge. No soluble 
pigment. Hydrolysis good. 

Cellulose: No growth. 

Nitrate reduction: None. 

Production of H.S: Positive. 

Carbon utilization: Sucrose, mannose, dex- 
trin, galactose, glycerol, fructose, glucose, 
maltose, mannitol, xylose, and sodium suc- 
cinate utilized. Arabinose, esculin, rhamnose, 
dulcitol, sodium acetate, mulin, lactose, sali- 
cin, and raffinose not utilized. 

Antagonistic properties: Produces an anti- 
biotic active against mycobacteria and iden- 
tical with actithiazice acid or thiozolidone. 

temarks: The culture resembles S. roseo- 
chromogenes in color of growth and in no or 
slow liquefaction of gelatin. It differs in the 
lack of spiral formation and of nitrate re- 
duction. Gause et al. (1957) described a 
variety of this organism under the name of 
A. cinnamonensis var. proteolyticus. A. dag- 
and A. fumanus described by 
these authors apparently also belong to this 


hestanicus 


group, although they differ from it in some 
respects. According to Benedict and Prid- 
ham (1959) a group of cooperators consid- 
ered this organism as S. cinnamonensis, S. 
pirginiae, S. acidomyceticus, S. roseochromo- 
genes, and S. lavendulae; an opinion was ex- 
pressed that all of these are probably re- 
lated to S. lavendulae. 
Type culture: ATCC 12,308. 


THE ACTINOMYCETES, Vol. II 


59. Streptomyces circulatus (krassilnikov, 
1941) Waksman (Krassilnikov, N. A. Actino- 
mycetales. Izvest. Akad. Nauk. SSSR, Mos- 
kau, p. 60, 1941). 

Morphology: Sporophores produce verti- 
cils with spiral-shaped short branches. Spores 
cylindrical or oblong, 1.5 by 0.7 wu, some 
rounding up with age of culture. 

Synthetic agar: Growth good, colorless. 
Aerial mycelium abundant, white. 

Nutrient Growth weak. No aerial 
mycelium. 

Gelatin: Liquefaction weak. 

Milk: No coagulation; slow peptonization. 

Starch: Hydrolysis weak. 

Cellulose: No growth. 

Paraffin: Growth good. Aerial mycelium 


agar: 


white. 

Nitrate reduction: Weak. 

Sucrose: No inversion. 

Antagonistic properties: Limited. 

Habitat: Soil. 

56. Streptomyces citreus (Xrainsky, 1914) 
Waksman and Henrici, 1948 (Krainsky, A. 
Centr. Bakteriol. Parasitenk. Abt. IL, 41: 
684, 1914; Waksman, 8S. A. and Curtis, R. 
E. Soil Sci. 1: 116, 1916; 8: 121, 1919). Not 
Actinomyces citreus Gasperini, 1894. 

Morphology: Sporophores form long nar- 
row, open spirals, dextrorse. Spores spheri- 
cal to oval, 1.2 to 1.5 by 1.2 to 1.8 yp. 

Sucrose nitrate agar: Growth abundant, 
raised, wrinkled, citron-yellow. Aerial myce- 
lium white to citron-yellow. No soluble pig- 
ment. 

Malate-glycerol agar: 
vellow. Aerial mycelium white with mouse- 
gray tinge. No soluble pigment. 

Glucose-asparagine agar: Growth glossy, 
olive-yellow; center elevated. Aerial myce- 
lium white to pinkish. No soluble pigment. 

Nutrient agar: Growth restricted, green. 
No aerial mycelium. No soluble pigment. 

Potato: Growth yellowish to gray. Aerial 
mycelium white. No soluble pigment. 

Gelatin: Surface growth restricted, yellow- 


Growth creamy to 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


ish. Aerial mycelium white. Liquefaction me- 
dium. Melanin-negative. 

Milk: Surface growth cream-colored. Co- 
agulation followed by rapid peptonization. 

Starch media: Growth abundant, citron- 
yellow to yellowish-green. Aerial mycelium 
pinkish. Rapid hydrolysis of starch. 

Cellulose: No growth. 

Invertase: Positive. 

Nitrate: Sight reduction to nitrite. 

Production of HS: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Negative. 

Habitat: Garden soil. 

Remarks: Since Krainsky’s culture was 
not available for comparison, the above de- 
scription is based upon that of Waksman and 
Curtis (1916) and Waksman (1919); some dif- 
ferences exist between this description and 
that of Krainsky. Ettlinger et al. (1958) con- 
sidered this culture as a strain of S. griseus. 
KrassilInikov (1949) considers this organism 
as similar to Gasperini’s culture, both being 
looked upon as varieties of A. flavus. 

Type culture: IMRU 3574. 

57. Streptomyces clavifer (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores long, straight, 
some terminating in club-shaped structures. 
Spores cylindrical, 1.5 by 1.0 yp. 

Sucrose nitrate agar: Growth gray to 
brick-red. Aerial mycelium white, sprinkled 
with cinnamon-drab. Soluble pigment. yel- 
lowish to brown. 

Potato: Growth wrinkled, gray to orange 
to brown. Aerial mycelium gray to olive- 
buff. Color of plug gray to brown. 

Gelatin: Growth gray to buff. Aerial my- 
celium white. Medium liquefaction. Soluble 
pigment yellow to reddish-yellow. 

Starch: Hydrolysis. 

Tyrosinase reaction: Positive. 

Nitrate: No reduction. 

Temperature: Fair growth at 37.5°C. 


197 


Habitat: Limed soil and common scab of 
potatoes. 

temarks: Kutzner (1956) described the 
original culture obtained from CBS as pro- 
ducing an ash-gray aerial mycelium without 
spirals, and as melanin-negative; this culture 
was indistinguishable from S. craterifer also 
CBS. Krassilnikov (1949) 
considered it as a variety of A. scabies. 


obtained from 


58. Streptomyces coelicolor (Miiller, 1908) 
Waksman and Henrici emend. Kutzner and 
Waksman (Miller, R. Centr. Bakteriol. 
Parasitenk. Abt. I, Orig. 46: 195, 1908; 
Kutzner, H. J. and Waksman, S. A. 
J. Bacteriol. 78: 528-538, 1959). 

Synonyms: 

Streptothrix coelicolor Miller (Miller, 
1908). 

Actinomyces albidoflavus (strain Hohle, 
CBS). 

Actinomyces alni (strain v. Plotho, CBS). 
Streptomyces canescus Hickey et al. 
(Hickey et al., 1952, NRRL 2419). 

Possible synonyms: 
Gause et al. (Gause et al., 1957). 
KrassiInikov (Krassilnikov, 


A. cyaneofuscatus 


A. levoris 
1958). 

Not S. violaceoruber. 

Morphology: Sporophores of most strains 
short, arranged in small tufts, wavy; no 
spirals. Spores spherical to ellipsoidal; sur- 
face smooth. 

Agar media: Substrate growth on most 
media colorless or atypical yellowish-brown- 
ish; sometimes pinkish-red, especially in the 
lower part of the slants. Aerial mycelium 
colored grayish-yellow, often with a green- 
ish or pinkish shade. Soluble pigment on 
most media either absent or yellowish- 
brown. Blue pigment is produced by some 
strains on glucose-caletum malate-NHyNOs 
agar, mannitol-calcium malate-peptone agar, 
or glucose-peptone agar. 

Potato: Growth abundant, lichenoid. Aer- 
to yellow. 


ial mycelium powdery, white 


Characteristic formation of greenish-blue to 


198 


sky-blue soluble pigment by several strains; 
it may later become deep blue or blue-violet. 
Addition of glycerol delays pigment forma- 
tion. 

Gelatin: Good growth. Rapid liquefaction. 
No soluble pigment. 

Milk: No coagulation, rapid peptoniza- 
tion, complete within 15 days at 22-27°C; 
coagulation within 3 to 5 days, followed by 
peptonization at 36°C. 

Starch hydrolysis: Strong. 

Nitrate reduction: Positive; none reported 
for S. canescus by Hickey et al. (1952). 

Carbon sources: Utilizes L-xylose, L-arabi- 
nose, p-fructose, D-galactose, pb-mannitol, 
salicin; does not utilize L-rhamnose or raffi- 
nose; most strains do not utilize sucrose. 

Hemolysis of blood: Rapid at 37°C. 

Production of H.S: Negative. 

Antagonistic properties: Active upon sev- 
eral fungi and yeasts; all strains as far as 
tested produce polyene antibiotics. S. griseus 
(Krainsky) Waksman and Curtis (1916) 
probably belongs to this species, since it 1s 
now known to produce an antifungal agent 
of the polyene type. 

Ecology: S. coelicolor is widely distributed 
in nature. In a search for polyene-producing 
organisms, Pledger and Lechevalier (1955— 
1956) found 26 strains among 93. isolates 
which produced polyenes and which can be 
regarded as belonging to this species. Among 
the 382 subgroups of Kutzner (1956), the 
S. coelicolor subgroup was the one which 
comprised most Heymer (1957) 
found this organism strikingly often on the 
skin and in the tonsils of men. The first 
culture of this species isolated by Miller 
(1908) and the ascosin-producing organism 
GS. canescus) were found as chance con- 


strains. 


taminants; this indicates the wide distribu- 
tion of the organism in air. The relationship 
of blue pigment-forming bacteria, designated 
as Actinobacillus and Actinococcus, to this 
was Beijerinck 


organism discussed — by 


(1913a). 


THE ACTINOMYCETES, Vol. II 


Numerous cultures isolated by different 
investigators and described as S. coelicolor 
belong to S. violaceoruber. Others, however, 
such as A. tricolor Wollenweber, are related 
to S. coelicolor. 

Type culture: A strain of this organism 
was deposited by R. Miiller in the CBS. 


59. Streptomyces collinus Lindenbein, 1952 
Lindenbein, W. Arch. Mikrobiol. 17: 361— 
383, 1952). 

Morphology: 
Spores oval. 

Glycerol nitrate agar: Growth yellow- 
brown to red-brown. Aerial mycelium chalk- 
white. Soluble pigment yellow-brown, later 
becoming reddish-brown. 

Glucose-asparagine agar: Growth yellow- 
brown to purple-red. Aerial mycelium chalk- 
white, later ash-gray. Soluble pigment car- 
mine-red, later brown-red. 


Sporophores form. spirals. 


Glycerol malate agar: Growth  yellow- 
brown to red-brown. Aerial mycelium vel- 
vety, chalk-white. Soluble pigment yellow- 
brown. 

Nutrient agar: Growth dark brown. Aerial 
mycelium powdery, gray-white. Soluble pig- 
ment dark brown. Melanin-positive. 

Glucose-peptone agar: Growth  yellow- 
brown and red. Aerial mycelium velvety, 
white. Soluble pigment chestnut-brown. 

Starch media: Growth reddish to orange. 
Aerial mycelium white. Hydrolysis medium. 

Potato: Growth good. Aerial mycelium 
white. No soluble pigment. 

Gelatin: Growth dark brown. No aerial 
mycelium. Soluble pigment dark brown. 
Liquefaction rapid. 

Milk: Growth good; dark brown reverse. 
Aerial mycelium white, later ash-gray. Solu- 
ble pigment dark brown. No peptonization. 

Cellulose: Growth good, colorless. 

Antagonistic properties: Produces an anti- 
biotically active pigment. 

Habitat: Soil. 

Remarks: Closely related to S. erythro- 
chromogenes. Gause et al. (1957) described a 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


similar form under the name of A. albovina- 


ceus. 


60. Streptomyces coroniformis (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight, some 
long and some short. Spores oval, 0.8 by 
0.6 p. 

Sucrose nitrate agar: Growth in form of 
discrete colonies partially coalescing, gray to 
greenish. Aerial mycelium white, covering 
edges of growth. 

Nutrient potato agar: Growth wrinkled, 
grayish. No aerial mycelium. 

Potato: Growth raised, grayish. Aerial 
mycelium white. Plug pigmented brownish 
around and under growth. 

Gelatin: Growth fair. Liquefaction slow if 
any. 

Milk: A few colonies on surface. No coagu- 
lation; peptonization limited. 

Starch: No hydrolysis or trace. 

Nitrate: Limited reduction to nitrite. 

Tyrosinase reaction: Negative. 

Temperature: Growth fair at 37.5°C 

Habitat: Potato scab. 

61. Streptomyces craterifer (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Straight sporophores form 
terminal branches, dichotomously forked. 
Spores rectangular, 1.8 to 0.9 by 1.0 to 0.8 wu. 

Sucrose nitrate agar: Growth lichenoid, 
abundant, colorless; aerial mycelium mouse- 
gray. Numerous guttation drops, which 
leave blackish craters on surface of growth. 

Nutrient agar: Growth colorless; aerial 
mycelium scant, white. No soluble pigment. 

Starch agar: Growth spreading, thin, col- 
orless; no aerial mycelium. Starch hydro- 
lyzed. 

Potato: Growth cream-colored; aerial my- 
celium white to mouse-gray. Color of plug 
unchanged. 

Gelatin: Surface growth wrinkled; aerial 


199 
mycelium white. No 
soluble pigment. 

Milk: Surface growth cream-colored. No 
coagulation, rapid peptonization. 


Rapid liquefaction. 


Tyrosinase reaction: Negative. 

Nitrate reduction: Positive. 

Temperature: Only 
See 

Habitat: Raised, smooth scab. 

Type culture: IMRU 3373. 

62. Streptomyces cyaneus (Krassilnikoy, 
1941) Waksman (Krassilnikov, N. A. Actin- 
omycetales. Izvest. Akad. Nauk. SSSR, Mos- 
kau, p. 14, 1941). 

Morphology: Sporophores produce open 
spirals (sinistrorse), with 2 to 3 


shght growth at 


turns in 
ach. Spores oval, seldom spherical, 0.6 to 
0.8 by 0.6 uw. 

Agar media: Growth pigmented blue at 
both acid and alkaline reactions. The pig- 
ment does not dissolve into medium. Aerial 
mycelium well developed, downy, bluish- 
gray to blue-green in color. 
nitrate Colonies at first 
smooth, becoming lumpy, leathery, com- 
pact, and covered with well developed blue- 
gray aerial mycelium. 

Gelatin: Strong 
positive. 

Milk: Peptonization without prior coagu- 


Sucrose 


agar: 


liquefaction. Melanin- 


lation. 

Starch: Hydrolysis weak. 

Cellulose: No growth. 

Nitrate reduction: Negative. 

Sucrose inversion: Negative. 

Antagonistic properties: Weak. 

Type culture: IMRU 3761. 

63. Streptomyces cyanoflavus Funaki and 
Tsuchiya, 1958 (Funaki, M., Tsuchiya, F., 
Maeda, K., and Kamiya, T. J. Antibiotics 
(Japan) LLA: 143-149, 1958). 

Morphology: Sporophores straight, form- 
ing many branches, but no spirals. 

Sucrose nitrate agar: Growth colorless to 
pale yellowish-brown. Aerial mycelium white 
to light greenish-gray. No soluble pigment. 


200 THE ACTINOMYCETES, Vol. II 


Glucose-asparagine agar: Growth pale 
green to yellowish-brown. Aerial mycelium 
brownish-white to brownish-gray. Soluble 
pigment hght blue to yellowish-brown. 

Calcium malate agar: Growth light brown- 
ish to brown. Aerial mycelium white to gray. 
Soluble pigment hight 
brown. 

Nutrient agar: Growth yellowish-brown to 
brown. Aerial mycelium grayish-white. No 
soluble pigment. 

Yeast extract agar: Growth pale yellow to 
brown. Aerial mycelium light olive-gray. 
Soluble pigment brown. 

Potato: Growth yellow to brown. Aerial 


greenish-blue — to 


mycelium brownish-gray. Soluble pigment 
dark brown. 

Gelatin: Growth yellow. Soluble pigment 
yellow to brownish-yellow. Gelatin lique- 
fied. 

Milk: Produces a sedimented growth with- 
out any soluble pigment. Milk coagulated 
but not peptonized. 

Carbon utilization: Utilizes various sugars 
and salts of organic acids, but not xylose, 
acetate, or citrate. 

Antagonistic properties: Produces blue 
antibiotic ¢cyanomycin, active against gram- 
positive and gram-negative bacteria; also 
produces aureothricin-like substances. 


64. Streptomyces cylindrosporus (kXrassil- 
nikov, 1941) Waksman (Krassilnikoy, N. A. 
Actinomycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 57, 1941). 

Morphology: Sporophores straight, 
branched. Spores cylindrical or oblong, 1.0 
bowler. oy O57 jes 

Sucrose nitrate agar: Colonies velvety, 
dark brown or chocolate. Aerial mycelium 
white-gray to brown-gray. Soluble pigment 
brown. 

Nutrient agar: Growth dark brown. Aerial 
mycelium white. Soluble pigment brown. 

Glucose-asparagine agar: Growth brown. 
Aerial mycelium white-gray. No soluble pig- 
ment. 


Gelatin: Weak liquefaction. Melanin-posi- 
tive. 

Milk: Coagulation limited, peptonization 
weak; color of milk brown to almost black. 

Potato: Substrate growth brown. Aerial 
mycelium light gray. Soluble pigment brown. 

Starch: Weak hydrolysis. 

Cellulose: Limited, colorless growth. Aer- 
ial mycelium white. 

Nitrate reduction: Positive. 

Sucrose inversion: Negative. 

Antagonistic properties: None. 

Habitat: Soil. 

temarks: The description of the organism 
has been supplemented by Hoffmann (1958). 
It appears to be related to S. vinaceus, S. 
purpureochromogenes, and S. purpeofuscus. 
Gause et al. (1957) described a related form 
as A. wmbrinus. 

Type culture: IMRU 3764. 

65. Streptomyces diastaticus (Krainsky) 
Waksman and Henrici (Krainsky, A. Centr. 
Bakteriol. Parasitenk. Abt. II, 41: 682, 
1914). 

Morphology: Sporophores form tight spi- 
rals. Spores oval or spherical, 1.0 to 1.2 by 
LAstow5iueChies: 3738): 

Sucrose nitrate agar: Growth thin, gray. 
Aerial mycelium white, becoming drab gray. 

Calcium malate agar: Colonies 2 to 4 mm, 
yellowish when old. Aerial mycelium gray, 
with white outer zone; white specks fre- 
quently produced in gray mycelium. 

Glucose-asparagine agar: Growth yellow- 
ish, spreading. No aerial mycelium. 
Growth 
Aerial mycelium white, then gray. Soluble 


Nutrient agar: cream-colored. 
brown pigment. 

Potato: Growth white-gray. Aerial myce- 
lium gray and white. 

Gelatin: Liquefaction, with small, cream- 
colored flakes in liquefied part. 

Milk: Brownish ring. Coagulation and 
slow peptonization. 

Starch thin, colorless, 


agar: Growth 


—_—__ 


DESCRIPTION OF 


SPECIES OF STREPTOMYCES 201 


FrGuReE 37. Sporophores of S. diastaticus, X 4,500, showing uniform density over whole surface (Cour 


tesy of E. Baldacci, University of Milan, Italy). 


spreading. Aerial mycelium gray. Ready hy- 
drolysis. 

Cellulose: Good growth. 

Invertase: Negative. 

Nitrate reduction: Weak. 

Production of H.S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Limited. 

Habitat: Soil. 

temarks: This species was redescribed by 
Duché (1934) under the name A. 
ticus. Baldacci et al. (1955) raised this spe- 


roseodiasta- 


cies to the status of a ‘“‘series.’? Several new 
species or varieties were created: A. virido- 
diastaticus, A. diastaticus var. ardesicicus, A. 


diastaticus var. venezuelae, A. rubrocyano- 
diastaticus var. impiger and var. piger. 


Type culture: IMRU 3315. 


66. Streptomyces diastatochromogenes 
(Krainsky, 1914; Waksman and Curtis, 
1916) Waksman and Henrici, 1948 (krain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
IT., 41: 683, 1914). 


Morphology: According to Waksman and 
Curtis (1916), sporophores are straight. Ac- 
cording to Jensen (1930), sinistrorse spirals 
are produced. Spores spherical or oval, 1.2 yu. 
Growth 
Aerial 


later ash-gray. Soluble 


Sucrose nitrate colorless, 


agar: 


later yellowish-brown. mycelium 


abundant, white, 
pigment yellowish to light brown. 

Sucrose malate agar: According to IKrain- 
sky, growth colorless, with gray aerial myce- 
lium. When glucose is added, center of aerial 
mycelium is colored yellowish, with gray 
margin. 

Glucose-asparagine agar: Growth color- 
less, with gray aerial mycelium. 

Nutrient agar: Aerial mycelium white to 
gray. Soluble pigment brownish to coffee- 
brown. Melanin-positive. 

Potato: Growth light gray, later grayish- 
black. Aerial mycelium white to gray. Solu- 
ble pigment black. 

Gelatin: Growth cream-colored to vellow- 


ish-brown. Aerial mycelium scant white. Sol- 


202 


THE ACTINOMYCETES, Vol. II 


FIGURE 38. Sporophores of S. diastaticus, * 15,000 (Courtesy of E. Baldacci, University of Milan, 
Italy). 


uble pigment brown. Liquefaction fairly 


Habitat: Very common in soil. 
rapid. 


Remarks: Krassilnikov (1949) considered 
this species as a variety of A. chromogenes. 
Type culture: ATCC 12,309. 


Starch: Hydrolysis weak. 

Cellulose: No growth. 

Nitrate: Reduction to nitrite strong. 67. Streptomyces echinatus Corbaz et al., 
1957 (Corbaz, R., Ettlinger, L., G&asmann, 
Temperature: Optimum 35°C. E., Ixeller-Schierlein, W., Kradolfer, F., 
Antagonistic properties: Very strong. Neipp, L., Prelog, V., Reusser, P., and 


Tyrosinase reaction: Positive. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Zahner, H. Helv. Chim. Acta 40: 199-204, 
1957). 

Morphology: Sporophores produce verti- 
cils on sterile aerial hyphae, with open, ir- 
regular spirals. Spores elliptical to oval; sur- 
face of spores covered with long, thin spines 
(Pies). 

Glycerol nitrate agar: Growth greenish- 
yellow to citron-yellow to light green. Aerial 
mycelium white, changing to yellow, to ash- 
gray. Soluble pigment greenish-yellow to 
erass-green. 

Glycerol malate agar: Growth pale yellow, 
turning greenish-yellow. Aerial mycelium 
white to pale yellow. No soluble pigment. 

Glucose-asparagine agar: Growth golden 
vellow to greenish-yellow to greenish-gray. 
Aerial mycelium ash-gray to reddish-violet. 
No soluble pigment. 

Nutrient agar: Growth light yellow. No 
aerial mycelium. No soluble pigment. 
Glucose-peptone agar: Growth 
Aerial mycelium white in center, brownish 


yellow. 


on periphery, changing to ash-gray. Soluble 
pigment golden yellow. 

Starch agar: Growth yellow. Aerial myce- 
hum ash-gray. No hydrolysis, or at most, 
traces. 

Gelatin: Substrate growth dark brown. 
Aerial mycelium greenish-gray. Soluble pig- 
ment dark brown. No liquefaction after 31 
days. Melanin-positive. 

Potato: Growth greenish to raven-black. 
Aerial mycelium limited, white-gray to 
bluish-gray. Soluble pigment brownish to 
pitch-black. 

Milk: Good coagulation and peptoniza- 
tion. 

Tyrosinase reaction: Positive. 

Carbon utilization: Xylose, lactose, raffi- 
nose, acetate, and succinate—positive. Su- 
crose, inulin, duleitol, salicin, and sodium 
citrate—negative. 

Temperature: Develops well at 18-40°C. 

Antagonistic properties: Produces echino- 
mycin. 


205 


temarks: Closely related to S. griseoflavus 
and S. flaveolus. 


68. Streptomyces elasticus (Séhngen and 
Fol, 1914) Waksman (Séhngen, N. L. and 
Fol, J. G. Centr. Bakteriol. Parasitenk. Abt. 
IT, 40: 87-98, 1914). 

Morphology: Mycelium typical, branched. 
Short, motile rods observed in young cul- 
tures. Spores white, round, diameter about 
1 uw, double that of the mycelium. 

Agar Growth  yellowish-white. 
Aerial mycelium snow-white. 

Gelatin: Growth yellow-brown. 


media: 


Carbon utilization: Glucose, glycerol, ethyl] 
alcohol, mannitol, organic acids, 
salts readily assimilated. 


calelum 


Sucrose inversion: Positive. 

Urea: Produces urease. 

Paraffin: Utilized. 

tubber: Utilized readily. 

Temperature: Optimum 28°C, maximum 
33°C, destroyed at 65°C in 5 minutes. 

Habitat: Soil. 

69. Streptomyces endus Gottlieb and Car- 
ter, 1956 (Gottlieb, D. and Carter, H. E. 
U.S. Patent 2, 746, 902, May 22, 1956). 

Morphology: Sporophores formed along 
entire length of mycelium, at right angles 
to it. Compact spirals produced, often with 
10 loops. Young hyphae 0.7 to 1.0 uw in 
diameter; old hyphae 1.25 to 1.50 and even 
2.0) us. 

Sucrose nitrate agar: Substrate growth 
has color of medium, later turning dark. 
Aerial mycelium white, changing to light 
gray, then to dark gray. No soluble pigment. 

Gelatin: Slow and only slight liquefaction. 
No soluble pigment. Melanin-negative. 

Starch: Hydrolysis rapid. 

Potato: Growth good. Aerial mycelium 
hight gray. No soluble pigment. 

Milk: Coagulation; no visible peptoniza- 
tion. 

Carbon utilization: Utilizes starch, man- 


nose, dextrin, glucose, arabinose, maltose, 


204 


and levulose. Poor growth with galactose, 
lactose, citric acid, malic acid, succinic acid, 
and Does utilize 
sorbitol, duleitol, inositol, or paraffin. 


cellulose. not sucrose, 


Antagonistic properties: Produces an 
antibiotic, endomycin, active largely upon 
fung. 

Remarks: Tresner and Backus (1956) 


consider this organism as a variant of the 
S. hygroscopicus group. 


70. Streptomyces erythraeus  (Waksman 


and Curtis, 1916) Waksman and Henrici, 
1948 (Waksman, 8S. A. and Curtis, R. E. 
Soil Sci. 1: 99, 1916; Waksman, S. A. Soil 
sci: 8: 112, 1919). 

Morphology: Fine, monopodially 
branched aerial mycelium; numerous sporo- 
phores with open and closed spirals. Spores 
spherical to oval, 0.7 to 0.8 uh, smooth (PI. 
Il im). 

Sucrose nitrate agar: Growth yellowish, 
later becoming red. Pigment insoluble in 
medium. Aerial mycelium thick, white to 
pale rose. 

Glucose-asparagine agar: Growth abun- 
dant, spreading, cream-colored, later turn- 
ing brown chiefly on surface; center raised, 
lobate margin. 

Nutrient agar: Substrate growth cream- 
colored. No soluble pigment. 

Potato: Growth wrinkled, cream-colored, 
becoming yellowish to red to purplish. 
Melanin-negative. 

Gelatin: Growth abundant, dense, gray 
with pinkish tinge, chiefly on surface of 
slowly liquefied portion. No soluble pigment. 

Milk: Surface zone yellowish. Limited 
coagulation and positive peptonization. 

Starch media: Growth cream-colored with 
faint greenish tinge. Hydrolysis. 

Cellulose: Growth brick-red. 

Invertase: None. 

Nitrate: Reduction to nitrite only with 
starch. 

Production of HoS: Negative. 

Temperature: Optimum 25°C. 


THE ACTINOMYCETES, Vol. II 


Antagonistic properties: Marked. Pro- 
duces erythromycin A and B. 

Habitat: Soil. 

Remarks: According to Ettlinger et al. 
(1958) S. rimosus and S. roseochromogenes 
belong to this group. Krassilnikov (1949) 
considers this organism as a variety of A. 
ruber. A closely related, melanin-positive 
culture has been described as a new species, 
S. bottropensis (Brit. Pat. 762, 736, Nov. 19, 
1953). 

Type culture: IMRU 3737; ATCC 11,635. 

71. Streptomyces erythrochromogenes 
(Krainsky, 1914) Waksman and Henrici, 
1948 (kxrainsky, A. Centr. Bakteriol. Para- 
sitenk. Abt. II, 41: 679-682, 1914). 

Different strains of this organism have 
been studied by Krainsky (1914), Waksman 
and Curtis (1916), Jensen (1930), and Okami 
and Suzuki (1958). 

Morphology : Sporophores flexible, curved; 
spiral formation abundant according to 
Jensen and Okami and Suzuki. Waksman 
and Curtis reported no spirals. Spores oval. 
nitrate agar: Growth at first 
cream-colored, later turning red to violet to 
purple. Aerial mycelium white to light gray. 


Sucrose 


Soluble pigment red to red-violet according 
to Jensen. 

Calcium agar: Growth red to 
violet. Aerial mycelium grayish, with white 
margin. 


malate 


Glucose-asparagine agar: Aerial mycelium 
eray to white. Soluble pigment red. 

Nutrient agar: Growth yellowish-gray to 
light brown. Aerial mycelium white to light 
gray. Soluble pigment brown to deep brown. 

Starch agar: Aerial mycelium gray. Solu- 
ble pigment rose-colored. Diastatic action 
weak. 

Potato: Growth yellowish-gray, later al- 
most black. Aerial mycelium gray. Soluble 
pigment black. Melanin-positive. 

Gelatin: Growth yellowish to light purple. 
Liquefaction very slow. Soluble pigment 
brown. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Cellulose: Growth slow or none. 

Invertase: Negative. 

Nitrate: Reduction slight. 

Pigment: Soluble in water, not in organic 
solvents. 

Temperature: Optimum 380°C. 

Antagonistic properties: Produces sarko- 
mycin (Okami and Suzuki). 

Habitat: Not very common in soil. 

72. Streptomyces eurocidicus Okami et al., 
1954 (Okami, Y., Utahara, R., Nakamura, 
S.,and Umezawa, H. J. Antibioties (Japan) 
TA: 101-102, 1954). 


Morphology: Aerial mycelium — short, 
branched. Sporophores straight, without 


spirals; sometimes atypical verticils are 
produced. 

Glycerol nitrate agar: Growth colorless 
to yellowish-brown. Aerial mycelium scant, 
thin, white. No soluble pigment. 

Glucose-asparagine agar: Growth colorless 
to vellowish-brown. Aerial mycelium white 
with yellowish tinge. Soluble pigment absent 
or slightly brown. 

Nutrient agar: Growth vellowish-brown 
to black. Soluble pigment brown. Melanin- 
positive (?). 

Starch: Good hydrolysis. 

Potato: Growth wrinkled, brownish-yel- 
low. Aerial mycelium absent or thin white. 
No soluble pigment. 

Gelatin: Growth yellowish-brown. Soluble 
pigment brown. No liquefaction. 

Milk: Surface ring vellowish-brown. 

Nitrate: No reduction. 

Tyrosinase: Doubtful. 

Antagonistic properties: Produces an 
antifungal substance, eurocidin, and anti- 
bacterial substances tertiomycin and azomy- 
cin. 


73. Streptomyces eurythermus Corbaz et al., 
1955 
K., Keller-Schierlein, W., Neipp, L., Prelog, 
V., Reusser, P., and Zihner, H. Helv. Chim. 
Acta 38: 1202-1209, 1955). 


(Corbaz, R., Ettlinger, L., Gaiumann, 


205 


Morphology: Substrate growth consists 
of long hyphae. Aerial mycelium abundant, 
gray. Sporophores broom-shaped. Spores 
egg-shaped to spherical, smooth, 0.8 to 1.0 
by 0.6 to 0.7 u. 

Glycerol nitrate agar: Growth postulate 
light brown. Aerial mycelium sparse, white- 
gray, changing to ash-gray. Soluble pig- 
ment brown. 

Nutrient agar: Growth brownish-yellow. 
Aerial mycelium ash-gray. Soluble pigment 
reddish-brown. 

Glucose-asparagine agar: Growth thin, 
whitish yellow. Aerial mycelium white-gray 
to ash-gray. Soluble pigment chestnut- 
brown. 

Starch agar: Growth golden yellow. Aerial 
mycelium velvety, at first snow-white, later 
gray. Soluble pigment light brown. Rapid 
hydrolysis. 

Gelatin: Growth sparse. Soluble pigment 
dark brown. Rapid liquefaction. 

Potato: Growth — lichenoid, 
yellow. Aerial mycelium milky-white, be- 


brownish- 


coming ash-gray. Soluble pigment brownish 
to pitch-black. 

Milk: Brown surface ring. Aerial mycelium 
ash-gray. Soluble dark 
Coagulation and peptonization positive. 


pigment brown. 


Carbon utilization: Xylose, arabinose, 
fructose, galactose, saccharose, maltose, 
lactose, mannitol, salicin well utilized. 


Rhamnose, inulin, sorbitol, dulcitol, meso- 
inositol not utilized. Some strains use acetate, 
citrate, and succinate. 

Temperature: Poor growth at 18°C; very 
good growth at 30°C; good growth but no 
aerial mycelium at 58°C. 

Antagonistic properties: Produces a basic 
antibiotic, angolamycin, related to erythro- 
mycin. 

Habitat: Soil. 
Closely anti- 


temarks: related to S. 


bioticus. 


74. Streptomyces exfoliatus (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 


206 


(Waksman, 8. A. and Curtis, R. E. Soil Sei. 
PetG6; AGI6 S82 1211919). 

Morphology: Colony has tendency to 
crack and surface growth to exfohate and 
peel off. Sporophores usually straight or 
slightly wavy; on some media there is a 
tendency to produce spirals. Spores oval, 1.0 
tomleorbyalk2sto-lcSin- 


Sucrose nitrate agar: Growth smooth, 
colorless, becoming brown to blue. Aerial 
mycelium white. 

Malate-glycerol agar: Growth  cream- 


colored. Aerial mycelium white. No soluble 
pigment. 

Glucose-asparagine agar: Growth cream- 
colored, turning brown. Aerial mycelium 
white, appearing late. 

Nutrient agar: Growth No 
aerial mycelium. Soluble pigment absent or 


colorless. 


brownish. 
Potato: Growth wrinkled, gray, becoming 
brown. No aerial mycelium. No soluble pig- 


ment. 
Gelatin: Growth cream-colored. Aerial 
mycelium white or absent. Liquefaction 


faint to fair. Melanin-negative. 

Milk: Cream-colored ring. Soft coagula- 
tion and slow peptonization. 

Starch media: Growth restricted, gray 


becoming brown. Aerial mycelium light 
buff-gray. Hydrolysis of starch medium, 


incomplete. 

Invertase: Positive. 

Cellulose: Growth good. 

Nitrate reduction: Positive. 

Production of H.S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Positive. 

Habitat: Soil. 

femarks: Krassilnikov (1949) considered 
this organism as a variety of one of the 
chromogenic groups. 

Type culture: IMRU 3316. 

75. Streptomyces felleus Lindenbein, 1952 
(Lindenbein, W. Arch. Mikrobiol. 17: 361- 


383, 1952). 


THE ACTINOMYCETES, Vol. II 


Morphology: Sporophores long, straight, 
branching. Spores spherical, smooth. 


Glycerol nitrate agar: Growth smooth, 


o 
yellow-brown. Aerial mycelium velvety 
eray-white. Soluble pigment —yellowish- 


brown. 

Glucose-asparagine agar: Growth colorless 
to brownish-yellow. Aerial mycelium gray- 
white. Soluble pigment brownish. 

Glycerol malate agar: Growth 
to yellowish. Aerial mycelium powdery, 
eray-white. Soluble pigment 
brown. 

Nutrient 
ish-yellow 
Soluble pigment 
Melanin-negative. 

Glucose-peptone agar: Growth yellowish- 
brown. Aerial mycelium gray-white. Soluble 
pigment light brown. 

Starch media: Growth lichenoid, colorless. 
Aerial mycelium white. No soluble pigment. 
Hydrolysis strong. 


colorless 
yellowish- 
agar: Growth colorless, brown- 


reverse. No aerial mycelium. 
light brownish-yellow. 


Potato: Growth  brownish-yellow. No 
aerial mycelium. Soluble pigment absent or 
pinkish. 


Gelatin: Growth colorless. No aerial myce- 
lium. No soluble pigment. No lquefaction 
by one strain, positive by another. 

Milk: Growth brownish to orange. Aerial 


mycelium gray-white. Peptonization me- 
dium. 
Cellulose: No or weak growth. 
Production of HS: Negative. 
Odor: Typical earthy. 
Taste: Gall-bitter. 
Antagonistic properties: Produces anti- 


biotic picromycin. 

Remarks: Related to S. fimicarius. Ett- 
linger et al. (1958) considered this organism 
as belonging to S. olivaceus. 

Type culture: IMRU 3659. 

76. Streptomyces fervens DeBoer et al., 
1959 (DeBoer, C., Dietz, A., Evans, J. 8., 
and Michaels, R. M. Antibiotics Ann. 1959- 
1960, pp. 220-226). 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Morphology: Sporophores monoverticil- 
late or biverticillate. Pigment granules 
present in mycelium. 

Sucrose nitrate agar: Growth faint-pink. 
Aerial mycelium pink. No soluble pigment. 

Calcium malate agar: Growth © pink. 
Aerial mycelium trace, pink. No soluble pig- 
ment. 

Glucose-asparagine 
Aerial mycelium pink. Soluble pigment pale 


agar: Growth pink. 


yellow. 

Starch nutrient agar: Growth red-pink. 
Aerial mycelium pink. Soluble pigment tan. 

Casein digest-beef extract agar: Growth 
red. Aerial mycelium pink. 

Gelatin: Liquefaction medium. Soluble 
pigment brown. 

Milk: Growth cream-pink. 
Aerial mycelium trace pink. No coagulation. 
No peptonization. 

Production of H.S: Positive. 

Starch: Hydrolyzed. 

Carbon utilization: Utilizes various carbo- 
hydrates, glycerol, inositol, starch, certain 


brown to 


organic acids (acetate, citrate, succinate) ; 
does not utilize d-xylose, rhamnose, lactose, 
l-arabinose, formic, oxalic, and tartaric acids. 

Antagonistic properties: Produces anti- 
biotic fervenulin, active against various 
microbes and tumors. 


Habitat: Soil in California. 


77. Streptomyces filamentosus Okami et al., 
1953 (Okami, Y., Okuda, T., Takeuchi, T.., 
Nitta, K., and Umezawa, H. J. Antibiotics 
(Japan) 6A: 153-157, 1953). 

Morphology: Sporophores straight, long, 
without spirals. Spores oval to elliptical. 

Sucrose nitrate agar: Growth colorless. 
Aerial mycelium abundant, cottony, white. 
No soluble pigment. 

Glucose-asparagine agar: Growth color- 
less. Aerial mycelium abundant, white with 
pinkish-orange tinge or brownish to almost 
salmon-pink tinge. No soluble pigment. 

Nutrient agar: Growth colorless. Aerial 
mycelium thin, white. No soluble pigment. 


207 


Starch agar: Growth same as on synthetic 
agar. Aerial mycelium white or white with 
light brownish-salmon-pink tinge. Hydrol- 
ysis. 

Gelatin: Growth yellowish. Aerial myce- 
hum in form of white patches. No soluble 
pigment. Medium liquefaction. 

Potato plug: Growth cream-colored, wrin- 
kled. No aerial mycelium. No soluble pig- 
ment. 

Milk: Growth yellowish, surface 
Aerial mycelium white, scant. No soluble 
pigment. Coagulation and peptonization. 

Blood agar: Growth brownish, wrinkled. 
No aerial mycelium. Hemolysis none or 
weak. 

Antagonistic properties: Produces caryo- 
mycin, an antitumor substance. 


ring. 


78. Streptomyces filipinensis Ammann et 
al., 1955 (Ammann, A., Gottheb, D., Brock, 
T. D., Carter, H. E., and Whitfield, G. B. 
Phytopathology 45: 559-563, 1955). 

Morphology: Sporophores form 
that vary from open to tightly closed. Spores 


spirals 


round to oval. 

Sucrose nitrate agar: Growth excellent, 
hight Aerial mycelium 
white, turning gray. Soluble pigment slightly 


yellow. cottony, 


yellow. Colorless drops of exudate on myce- 


lium. 
Starch-nitrate agar: Growth excellent. 
Aerial mycelium velvety, white, turning 


gray. No soluble pigment. Hydrolysis weak. 

Glycerol-asparagine agar: Growth excel- 
lent. Aerial mycelium white, turning gray. 
Soluble pigment slightly yellow. 

Nutrient agar: Growth very poor, light 
buff. No aerial mycelium. Soluble pigment 
brown. 

Gelatin: Slow but definite liquefaction, 
stratiform type. Soluble pigment brown. 

Potato: Growth good. No aerial mycelium. 
Soluble pigment purple to black. 

Nitrate reduction: Little, if any. 

Production of H.S: Positive. 

Carbon utilization: Utilizes xylose, arabi- 


208 


nose, fructose, galactose, sucrose, maltose, 
lactose, raffinose, mnulin, mannitol, imositol, 
sodium acetate, sodium citrate, sodium 
succinate, dextrose, mannose, starch, dex- 
trin, and glycerol. Does not utilize rhamnose, 
sorbitol, dulcitol, salicin, phenol, m-cresol, 
sodium formate, sodium oxalate, sodium 
tartrate, or sodium salicylate. 

Antagonistic properties: Produces anti- 
fungal agent filipin, of the polyene type. 

Habitat: Philippine soil. 

Type culture: IMRU 3781. 


79. Streptomyces fimbriatus (Millard and 
Burr, 1926) Waksman and Henrici, 1948 
(Millard, W. A. and Burr, 8. Ann. Appl. 
Biol. 13: 580, 1926). 

Morphology: Sporophores form spirals 
with three or more turns. Spores cylindrical 
to oval, 1.2 to 0.9 by 0.9 uz. 

Sucrose nitrate agar: Growth gray. Aerial 
mycelium abundant, white to gray. Soluble 
pigment cream-colored. 

Glucose-asparagine agar: Growth 
good. Aerial mycelium white to mouse-gray. 

Nutrient potato agar: Colonies gray to 
blackish, flat, raised in center. Aerial myce- 
lium a few specks of white. Soluble pigment 


very 


golden brown. 

Potato: Growth mouse-gray. Aerial myce- 
lium on dried portions of growth secant, white 
to mouse-gray. Pigment around growth 
black. 

Gelatin: Growth good. Aerial mycelium 
white. Liquefaction slow. Soluble pigment 
reddish. 

Milk: Growth good. No coagulation and 
no hydrolysis. 

Starch: Positive hydrolysis. 

Tyrosinase reaction: Strongly positive. 

Nitrate reduction: Positive. 

Habitat: Common scab of potatoes. 

80. Streptomyces fimicarius (Duché, 1934) 
Waksman and Henrici, 1948 (Duché, J. 
actinomyces du groupe albus. P. Lechevaler, 
Paris, 1934). 


Les 


THE ACTINOMYCETES, Vol. II 


Morphology: Sporophores long, tuft- 
forming; no spirals. Spores cylindrical (Hoff- 
mann, 1958). 

Sucrose nitrate agar: Growth at first color- 
less, later yellowish to red-brown; reverse 
orange-colored. Aerial mycelium light gray 
with yellowish tone. Soluble pigment faint 
yellowish. 

Glucose-asparagine agar: Growth cream- 
colored with whitish aerial mycelium; re- 
verse cream-colored to slight ocher. 

Nutrient agar: Growth limited, cream- 
colored with white aerial mycelium; reverse 
yellowish. 

Potato: Growth cream-colored to yellow- 
ish to dark brown. Aerial mycelium gray. 
Soluble pigment reddish-brown. 

Gelatin: Punctiform colonies with whitish 
aerial mycelium. Soluble pigment reddish. 
Liquefaction medium. Melanin-negative. 

Milk: Growth colorless, becoming covered 
with whitish aerial mycelium. Slow  pep- 
tonization. Pigment rose, changing to brown- 
ish-red. 

Starch: Hydrolyzed. 

Coagulated serum: Growth cream-colored. 
Aerial mycelium whitish. Liquefaction rapid. 

Cellulose: No growth. 

Tyrosine medium: Growth white, with 
yellowish reverse. Soluble pigment yellowish. 

Production of HoS: Negative. 

Antagonistic properties: Positive. 
Krassilnikov (1949) 
this organism as a variety of A. chromogenes. 


81. Streptomyces flaveolus | (Waksman) 
Waksman and Henrici, 1948 (Waksman, 
S. A. No. 168. Soil Sci. 8: 134, 1919). 

Morphology: Sporophores monopodially 
branched. Short, closed and open spirals 


temarks: considers 


produced on all media. Spores oval to 
elliptical, 0.8 by 1.2 uw, covered with long, 
fine hair. 

Sucrose nitrate agar: Growth light sulfur- 
cadmium-yellow. Aerial 
to ash-gray. Soluble pig- 


vellow, turning 
mycelium white 
ment yellow. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Malate-glycerol agar: Growth colorless to 
cream-colored. Aerial mycelium mouse-gray. 
Glucose-asparagine agar: Aerial mycelium 
pale gray. Soluble pigment yellowish-green. 
Nutrient agar: Growth colorless, glisten- 
ing, wrinkled. Aerial mycelium white. Solu- 
ble pigment absent or yellow. 
Potato: Growth abundant, 
cream-colored to yellow. Aerial mycelium 
white to pinkish. Soluble pigment absent or 


wrinkled, 


faint brown. 
Gelatin: 

spreading. Aerial mycelium white. Liquefac- 

tion rapid. Soluble pigment vellowish-brown, 


Growth abundant, yellowish, 


not melanoid. 

Milk: Ring sulfur-yellow. Rapid coagula- 
tion and strong peptonization. 

Starch media: Growth colorless. Aerial my- 
celium light gray. Hydrolysis. 

Cellulose: Growth scant. 

Invertase: Negative. 

Nitrate reduction: Positive. 

Production of HoS: Negative. 

Antagonistic properties: Some — strains 
produce actinomycin. 

Habitat: Soil. 

Remarks: Several varieties of this organ- 
ism have been described. It is sufficient to 
mention a culture described by Krassilnikov 
as A. rectus, which appears to be a variety 
of S. flaveolus. WrassilInikov also believed 
that A. krainskia Duché belongs to this 
group. 

Type culture: IMRU 3319. 

82. Streptomyces flavochromogenes (Krain- 
sky, 1914) Waksman Henrici, 1948 
(Krainsky, A. Centr. Bakteriol. Parasitenk. 
Abt. II, 41: 685, 1914). 

Morphology: Spores oval, 1.7 wu. 


and 


Glucose-asparagine agar: Growth yellow. 


Aerial mycelium gray. Soluble pigment 
brown. 

Calcium malate agar: Growth yellow. 
Aerial mycelium produced late, white to 
oray. 


Nutrient agar: Aerial mycelium formed 


209 


late, at first white, later gray. Soluble pig- 
ment brown. 

Starch agar: Growth yellow. Aerial myce- 
hum white. Weakly diastatic. 

Potato: Growth yellow. Aerial mycelium 
white. Soluble pigment black. 

Gelatin: Colonies yellowish. Slight lique- 
faction. Soluble pigment brown. 

Cellulose: Growth slow. 

Nitrate reduction: Strong. 

Tyrosinase: Positive. 

Temperature: Optimum 

Habitat: Garden soil. 

temarks: Krainsky considered this cul- 


Bonu: 


ture as identical to A. chromogenes Gas- 
perini. 
Type culture: IMRU 3671. 


85. Streptomyces — flavogriseus — (Duché, 
1954) Waksman (Duché, J. Les actinomyces 
du groupe albus. P. Lechevalier, Paris, 1934). 

Morphology: Sporophores long, straight, 


with a few curling tips. Spores spherical. 


Sucrose nitrate agar: Growth limited, 
yellowish, reverse turning black. Aerial 


mycehum thin, gray to mouse-gray. 
Nutrient agar: thin, cream- 
colored. Aerial mycelium thin, white. No 


Growth 


soluble pigment. Melanin-negative. 


Glucose-peptone agar: Growth yellow; 
reverse tending to turn dark. Aerial myce- 
hum abundant, drab. No 
soluble pigment. 


Starch agar: Growth very limited, similar 


mouse-gray to 


to that on sucrose nitrate agar. Hydrolysis. 


Potato: Growth abundant, lichenoid. 
Aerial mycelium abundant, mouse-gray to 
drab with white edge. No soluble pigment. 

Gelatin: Growth flocculent, through me- 
dium. Liquefaction slow. No soluble pig- 
ment. 

Milk: Cream-colored ring. No aerial myce- 
hum. Peptonization very rapid. 

femarks: According to Ettlinger et al. 
(1958) the S. 


this organism belongs to 


fradiae group. 


Type culture: IMRU 3322. 


210 


84. Streptomyces flavoreticult Funaki et al., 
1958 (Funaki, M., Tsuchiya, F., Maeda, 
K., and Kamiya, T. J. Antibiotics (Japan) 
LLA: 138-142, 1958). 

Morphology: Aerial mycelium is long with 
many short branches; numerous — small 
verticils are produced depending on the 
nature of the medium, especially on starch- 
ammonium agar. 

Sucrose nitrate agar: Growth colorless to 
pale yellow. Aerial mycelium white, cottony. 
Soluble pigment faint yellow. 

Glucose-asparagine agar: Growth pale 
yellow to yellowish-brown. Aerial mycelium 
yellowish-white to olive-gray. Soluble pig- 
ment pale yellow. 

Calcium malate agar: Growth pale yellow. 
Aerial mycelium pale yellow. No soluble 
pigment. 

Nutrient agar: Growth yellow to brown. 
Aerial mycelium pale yellow to yellowish- 
gray. Soluble pigment brown. 

Potato: Growth yellow, folded. Aerial 
mycelium yellowish white to olive-gray or 
olive-yellow. Soluble pigment dark brown. 

Milk: Colorless pellicle. Aerial mycelium 
white. Soluble pigment brown. Milk coagu- 
lated, then peptonized. 

Gelatin: Growth yellowish-brown. Aerial 
mycelium white to light gray. Soluble pig- 
ment brown. Gelatin liquefied. 

Starch: Not hydrolyzed. 

Antagonistic properties: Produces anti- 
biotic virocidin, which possesses antiviral and 
antibacterial properties. 

Xemarks: Similar to S. reticula and to S. 
flavus, differing from the first by the forma- 
tion of yellow growth and yellow soluble 
pigment and from the second by the forma- 
tion of verticils. 

85. Streptomyces flavovirens (Waksman, 
1919) Waksman and Henrici, 1948 (Waks- 
man, 8. A. Soil Sci. 8: 117, 1919). 

Morphology: Sporophores coarse, straight, 
large 


and short, relatively unbranched; 


masses of minute tufts; open spirals may be 


THE ACTINOMYCETES, Vol. II 


produced in certain substrates. Spores 
spherical, oval to rod-shaped, 0.75 to 1.0 by 
IO Mon 125: pa: 

Sucrose nitrate agar: Growth yellowish 
with greenish tinge. Aerial mycelium gray. 
Soluble pigment greenish-yellow. 

Glucose-asparagine agar: Growth — re- 
stricted, developing only to a very small 
extent into the medium, yellow, turning 
black. Soluble pigment golden yellow to 
greenish-yellow. 

Nutrient agar: Growth yellowish; reverse 
dark in center with yellowish zone and 
outer white zone. 

Potato: Growth sulfur-yellow, wrinkled. 

Gelatin: Surface pellicle yellowish-green. 
Good liquefaction. Melanin-negative. 

Milk: Cream-colored to brownish ring; 
coagulation and peptonization. 

Starch agar: Growth greenish-yellow, 
spreading, developing deep into the medium. 
Good hydrolysis. 

Invertase: Negative. 

Nitrate reduction: Limited. 

Production cf HS: Negative. 

Cellulose: No growth. 

Antagonistic properties: Produces actino- 
mycin. 

Habitat: Soil. 

temarks: Certain forms belonging to this 
species, such as A. griseostramineus and A. 
olivaceoviridis, have been described by Gause 
et al. (1957). Ettlinger et al. (1958) considers 
this species as belonging to the S. fradiae 
group. Hirsch (1960) considers this organism 
as an oligonitrophilic form. 

Type culture: IMRU 3320. 

86. Streptomyces flavus (Xrainsky, 1914) 
Waksman and Henrici, 1948 (kXrainsky, A. 
Centr. Bakteriol. Parasitenk. Abt. II, 41: 
685, 1914; Waksman, 8S. A. and Curtis, 
R. E. Soil Sei. 1: 99, 1916; 8: 71, 1919). 

Not A. flavus WKrainsky emend. Krassil- 
nikov (1941). 

Morphology: Sporophores are long, usu- 


== eee 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


ally no spirals; some open spirals may be 
produced. Spores oval, 1.2 yu. 

Sucrose nitrate agar: Growth yellow or 
sulfur-yellow. Aerial mycelium straw-yellow. 

Glucose-asparagine agar: Growth sulfur- 
yellow, center shading to brown. Aerial 
mycelium white to gray. 

Nutrient agar: Growth gray, spreading, 
folded. Aerial mycelium white, appears late. 

Starch agar: Growth cream-colored with 
pink tinge. Hydrolysis marked. 

Potato: Growth yellow. Aerial mycelium 
gray. Melanin-negative. 

Gelatin: Growth in form of small, yellow- 
ish masses on surface. Rapid liquefaction. 
Melanin-negative. 

Milk: Rapid coagulation and peptoniza- 
tion. 

Sucrose inversion: Negative. 

Nitrate: No reduction. 

Cellulose: Growth poor. 

Temperature: Optimum 25°C. 


Antagonistic properties: Some strains 
produce actinomycin and certain other 
antibiotics. 


Habitat: Soil. 

Remarks: Represents a large group of 
species, as shown previously (Chapter 3). 
Above description is based largely upon that 
given by Krainsky. According to Ettlinger 
et al. (1958), this organism does not form 
any spirals (as found also by Waksman and 
Curtis) and is related to S. olzvaceus. 

Type culture: IMRU 3321. 

87. Streptomyces flocculus (Duché, 1934) 
Waksman and Henrici (Duché, J. 
actinomyces du groupe albus. P. Lechevalier, 
Paris, 1934). 
nitrate 


Les 


Sucrose agar: Growth cream- 


later covered with white aerial 


mycelium. No soluble pigment. 


colored, 


Glucose-asparagine agar: Growth limited, 
cream-colored, only slightly raised above the 
surface of the medium; occasionally abun- 
dant growth produced with white aerial 
mycelium, colorless on reverse side. 


PA 
Nutrient agar: Growth cream-colored, 
later covered with white aerial mycelium. 
No soluble pigment. 

Potato: Growth punctiform. Aerial myce- 
lum white. Soluble pigment faint, yellowish. 

Gelatin: Growth limited. 
slow. Melanin-negative. 


Liquefaction 


Milk: Growth rose-colored. Peptonization 
slow. 

Coagulated serum: Growth cream-colored. 
Aerial mycelium fine, white. Liquefaction 
slow. 

Tyrosine medium: Growth whitish. No 
soluble pigment. 

Production of HS: Negative. 

Remarks: Belongs to the S. albus series. 


88. Streptomyces fradiae (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 
(Waksman, 8. A. and Curtis, R. E. Soil Sei. 
1: 99-134, 1916; 8: 90, 1919). 

Morphology: | Sporophores — branched 
monopodially, straight or flexible, but no 
true spirals. On certain media, such as 
glycerol agar, spirals are formed. Ettlinger 
et al. (1958) found open spirals. Spores oval 
to rod-shaped, 0.5 by 0.7 to 1.25 uw, smooth 
(Fig. 39). 

Sucrose nitrate agar: Growth smooth, 
spreading, colorless, or pale yellow-orange. 
Aerial mycelium thick, cottony, seashell- 
pink. No soluble pigment. 

Malate-glycerol 
Aerial mycelium seashell-pink. 


agar: Growth orange. 


Glucose-asparagine Growth  re- 


stricted, glossy, buff-colored, lichenoid mar- 


agar: 


gin. Aerial mycelium appears late, seashell- 
pink. 

Nutrient agar: Growth restricted, yellow- 
ish, becoming orange-yellow to buff. No 
aerial mycelium. No soluble pigment. 

Potato: Growth restricted, orange-colored. 
Aerial mycelium white to rose or pink. Solu- 
ble pigment absent or faint brown. 

Gelatin: Growth dense, cream-colored to 
brownish. Aerial mycelium white. Gelatin 
liquefied. No soluble pigment. 


FiGuRE 39. Sporophores of S. fradiae (Prepared 
by H. Lechevalier of the Institute of Microbiol- 
ogy). 


Starch media: Growth spreading, colorless. 
Aerial mycelium seashell-pink. Good dia- 
static action. 


Milk: Cream-colored ring; coagulation 
and rapid peptonization. 
Nitrate: Varied reduction. 


Cellulose: No growth in solution, fair 
growth on plates. 

Production of H.S: Negative. 

Invertase: None. 

Antagonistic properties: Highly antag- 
antibacterial agent, 
neomycin, and an antifungal agent, fradicin. 

Habitat: Soil. 

temarks: A 


onistic. Produces an 


this 
organism have been isolated from various 


number of strains. of 
soils (see, for example, S. decaris described as 
No. 3719, in Waksman et al., 1958). Some 
vary in their pigmentation, rate of gelatin 
liquefaction, and antibiotic production; A. 
longissimus IKrassilnikov is one such typical 
strain. Some strains are able to produce 
antiviral substances, as In the case of luridin, 
produced by a strain of S. fradiae designated 
as A. luridus by Wrassilnikov et al. Gause 
et al. (1957) described a_ spiral-producing 
variety of S. fradiae under the name spiralis. 
Several other such strains have been isolated 
by Waksman and Lechevalier, Umezawa, 
and many others. Ettlinger et al. (1958) 
claimed that S. 


rochei, S. filipinensis, S. 


2 THE ACTINOMYCETES, Vol. II 


coelicolor, S. flavogriseus, S. tyrosinaticus, S. 
violaceus, and S. violaceoruber belong to this 
group; this claim cannot be accepted on the 
basis of evidence submitted in the descrip- 
tions of these organisms. The characteristics 
of the species are that it is nonchromogenic, 
strongly proteolytic, and produces the 
characteristic seashell-pink aerial mycelium 
on various synthetic media; on organic 
media, orange-colored growth is produced 
without any aerial mycelium. 


Kor 


Type culture: IMRU 3535. 


89. Streptomyces fragilis Anderson et al., 
1956 (Anderson, L. E., Ehrlich, J., Sun, 
S. H., and Burkholder, P. R. Antibiotics & 
Chemotherapy 6: 100, 1956). 

Morphology: Aerial hyphae simple or 
branched, usually in small clusters; short, 
straight, or slightly curved, with bent or 
curved tips and occasional short spirals. 
Spores spherical to ovoid, 0.8 to 1.5 by 1.0 
to 2.0 p. 

Calcium malate agar: 
Aerial mycelium light brown. 


Growth sparse. 

Glycerol-asparagine agar: Growth sparse, 
colorless to light yellow. Aerial mycelium 
hight yvellow-pink. 

Starch-ammonium sulfate agar: Growth 
vellow to yellow-orange to orange-brown. 
Aerial mycelium light vellow-pink to lght 
brown. 

Nutrient agar: Growth yellow to yellow- 
orange to brown. Aerial mycelium white to 
light yellow-pink. Melanin-negative. 

Glucose-tryptone agar: Growth yellow to 
yellow-orange to brown. Aerial mycelium 
light vellow-pink, occasionally pink to gray- 
pink. 

Gelatin: Liquefaction slow. No soluble 
pigment. 

Litmus milk: Slow peptonization. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Carbon utilization: Utilizes L-arabinose, 
p-cellobiose, dextrin, b-galactose, glucose, 
p-maltose, starch, trehalose, and p-xylose. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Does not utilize aesculin, adonitol, cellulose, 
citrate, dulcitol, glycerol, 7-inositol, inulin, 
p-levulose, 
nose, melezitose, melibiose, pb-raffinose, L- 


p-lactose, D-mannitol, b-man- 
rhamnose, salicin, p-sorbitol, succinate, or 
sucrose. 

Antagonistic properties: Produces a sub- 
that 
anticancer properties. 

Source: Argentine soil. 

femarks: Closely resembles S. fradiae. 


stance, azaserine, possesses certain 


A detailed comparison between this and 
closely related organisms has been made by 
Anderson ef al. (1956). 

Type culture: IMRU 3732, NRRL 2424. 


90. Streptomyces  fulvissimus (Jensen, 
1930) Waksman and Henrici, 1948 (Jensen, 
H. L. Soil Sei. 30: 66, 1930). 

Morphology: Sporophores short, straight, 
often trifurcated. Slightly wavy, but no true 
spirals. Spores oblong, smooth, 1.0 to 1.2 
Dyaleatonh osu. blind). 

Sucrose nitrate agar: Growth light golden, 
later deep orange to red-brown. Aerial myce- 
lium scant, white, later grayish-brown. 
Soluble pigment bright golden to orange. 

Glycerol-asparagine agar: Growth golden 
to dark brown. Aerial mycelium white to 
light Soluble pigment 
golden to orange. 

Nutrient agar: Growth wrinkled, deep 


cinnamon-brown. 


red-brown. No aerial mycelium. Soluble pig- 

ment brownish-yellow. Melanin-negative. 
Potato: Growth wrinkled, 

Aerial mycelium absent or white. Soluble 


rust-brown. 


pigment gray to faint lemon-yellow. 
Gelatin: Growth yellowish-brown to red- 

brown. No aerial mycelium. No soluble pig- 

ment. Rapid liquefaction. 

agar: Growth 

brown. Aerial mycelium hydrolyzed, smooth, 

lead-gray. Soluble pigment dull yellow to 


Starch-caseim vellowish- 


orange starch. 

Production of HS: Positive. 

Antagonistic properties: Produces valino- 
mycin. 


Habitat: Very common in soil. 

Remarks: The characteristic golden pig- 
ment is formed in nearly all media, but 
becomes most typical and attains its greatest 
brightness in synthetic agar media. It has 
indicator properties, turning red in strongly 
acid solutions. The species is easily recog- 
nized on agar plates by its bronze-colored 
colonies, surrounded by halos of bright yel- 
low pigment. 

This species was believed to be identical 
with the culture described by Millard and 
Burr (1926) as A. flavus. The last name is 
invalid, however, since the culture could be 
readily distinguished from the S. flavus of 
Krainsky (1914, emend. Waksman 
Curtis, 1916) Waksman and Henrici. 

Type culture: IMRU 3665. 


and 


91. Streptomyces fumosus (Krassilnikoyv, 


mycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 58, 1941). 

Morphology: Sporophores straight. Spores 
cylindrical, later round, 1.5 to 2.0 by 0.7 u. 


Sucrose nitrate agar: Growth dark brown, 


pigment insoluble. Aerial mycelium well 
developed, cottony, dust-colored, occa- 


sionally gray-white. 

Nutrient agar: Growth dark brown. Aerial 
mycelium white. Soluble pigment brown. 

Potato: Aerial mycelium absent or only 
faint, dark gray. Melanin-negative. 

Gelatin: Liquefaction medium. 

Milk: No coagulation; slow liquefaction. 
Soluble pigment dark brown to almost black. 

Starch: Good hydrolysis. 

Cellulose: No growth. 

Sucrose: Inversion weak. 

Antagonistic properties: None 

Habitat: Soil. 

92. Streptomyces fungicidicus Okami et al., 
1954 (Okami, Y., Utahara, R., Nakamura, 
S., and Umezawa, H. J. Antibiotics (Japan) 
7A: 100-101, 1954). 

Morphology: Aerial mycelium produces 


214 


numerous spirals on most synthetic media. 
Spores spherical to oval. 

Glycerol nitrate agar: Growth colorless. 
Aerial mycelium white to grayish. 

Glucose-asparagine agar: Growth color- 
less. Aerial mycelium white to grayish. Solu- 
ble pigment sometimes yellowish. 

Calcium malate agar: Growth colorless to 
yellowish. Aerial mycelium white to grayish. 
Soluble pigment of some strains pink; later 
disappears. 

Nutrient agar: Soluble pigment absent or 
slightly yellowish-brown. Melanin-negative. 

Starch agar: Growth colorless to yellowish. 
Aerial mycelium white to grayish. Strong 
hydrolysis. 

Potato: Growth erayish. 
Aerial mycelium absent or white to grayish. 
A deep brown soluble pigment around the 
growth may be produced. 

Milk: Growth colorless to cream-colored. 
Soluble pigment absent or shghtly brown. 
Coagulation and peptonization weak. 

Gelatin: Growth colorless — to 
colored. Soluble pigment absent or faint 


yellowish to 


crealm- 


brown. Positive liquefaction. 
Nitrate: No, or doubtful reduction. 
Tyrosinase: Some strains positive. 
Antagonistic properties: Produces a poly- 
ene-type antifungal substance, fungicidin. 
femarks: Two groups of this species were 
recognized; Group A produces nonspiral- 
forming sporophores; aerial mycelium white; 
violet pigment on potato. It is thus differ- 
entiated from Group G, described above. 


93. Streptomyces fuscus (Sdhngen and Fol, 
1914) Waksman (Séhngen, N. L. and Fol, 
J. G. Centr. Bakteriol. Parasitenk. Abt. IT, 
40: 89-98, 1914). 

Morphology: According to Irassilnikovy, 
the organism forms short straight sporo- 
phores arranged in fascicles or clusters in the 
form of brushes. 

Agar media: Growth irregular, dry, color- 
less to stone-red. Aerial mycelium initially 
white, later becoming dark brown. Spores 


THE ACTINOMYCETES, Vol. II 


rose-colored. Some strains excrete a brown 
substance in protein media. 

Carbohydrates: Shght decomposition; 
even glucose is assimilated with difficulty. 

Carbon utilization: Best are 
calcium salts of various organic acids, rang- 
ing from malic and citric to stearate and 
palmitate. Formate not utilized. 

Nitrogen utilization: Ammonium chloride 
and asparagine, nitrate, and peptone assimi- 
lated with difficulty. 

Paraffins: Assimilated. 

Rubber: Brown-red growth. Rubber de- 
composed. 

Temperature: Optimum 33°C; maximum 
°C. Destroyed in 5 minutes at 65°C. 
Habitat: Soil. 


sources 


lod 


or 


94. Streptomyces galbus Frommer, 1959 
(Frommer, W. Arch. Mikrobiol. 32: 195, 


1959). 

Morphology: Sporophores monopodially 
branched, ending in spirals with 3 to 8 turns. 
On some media, certain strains produce long, 
straight, slightly branched aerial hyphae, 
with short side branches. 

Glycerol nitrate agar: Growth abundant; 
Aerial 
mouse-gray, or 


reverse yellow to yellow-green. 
mycelium 


green-gray. Soluble pigment golden yellow, 


cream-colored, 


later turning green-yellow. 
Glucose-asparagine agar: Growth weak, 
crusty, reverse light yellow or green-vellow, 
later turning brown. Aerial mycelium thin, 
powdery, with white spots. 
Soluble pigment yellowish to yellow-green. 
Calcium malate agar: Growth yellow to 
greenish-yellow. Aerial mycelium white to 
brownish-gray. Soluble pigment yellow to 


mouse-gray 


vellow-green. 
Nutrient agar: Growth thin, brown. No 
aerial mycelium. Soluble pigment brown. 
Starch-KNO; agar: Growth yellow. Aerial 
mycelium white to white-gray. Soluble pig- 
ment yellow. Slow hydrolysis of starch. 
Potato: Growth heavy, yellow to reddish- 
brown. Aerial mycelium powdery, white, 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


mouse-gray to green-gray. Soluble pigment 
black. 

Gelatin: Growth abundant. Aerial myce- 
lum yellow. Soluble pigment dark brown. 
Slow liquefaction. 

Milk: No coagulation; slow peptonization. 

Cellulose: Growth good. 

Antagonistic properties: Produces actino- 
mycin. 

Remarks: Related to S. vzridochromogenes, 
S. flavochromogenes, and S. viridoflavus. It 
was also said to be related to S. parvullus. A 
variety of this species designated as achro- 
mogenes, not producing any melanin pig- 
ment, was also described. 


95. Streptomyces galilaeus Ettlinger et al., 
1958 (Ettlnger, L., Corbaz, R., and Hiitter, 
R. Arch. Mikrobiol. 31: 356, 1958). 

Morphology: Sporophores monopodially 
branched, with open, regular spirals. Spores 
smooth (Pl. II k). 

Glycerol nitrate agar: Substrate growth 
at first light carmine, later carmine-red. 
Aerial mycelium white-gray. No soluble pig- 
ment. 

Glucose-asparagine agar: Growth thin, 
white-vellow, later red. Aerial mycelium 
ash-gray. No soluble pigment. 

Calcium malate agar: Growth thin, white- 
yellow, later red. Aerial mycelium ash-gray. 
No soluble pigment. 

Starch agar: Growth carmine-red. Aerial 
mycelium white-gray. Limited hydrolysis. 

Potato: Growth brownish-yellow. Aerial 
mycelium ash-gray. Soluble pigment limited, 
chestnut-brown. Melanin-positive. 

Gelatin: Surface growth light red to light 
brown. Aerial mycelium sparse, grayish- 
white. Soluble pigment reddish-brown to 
dark brown. Liquefaction trace. Melanin- 
positive. 

Milk: Pellicle thick, light brown. Aerial 
mycelium ash-gray. Limited coagulation, no 
peptonization. 

Antagonistic properties: Positive. 

Habitat: Soil. 


215 


96. Streptomyces galtiert (Goret and Jou- 
bert, 1951) Waksman (Goret, P. and Jou- 
bert, L. Ann. parasitol. humaine et comparée 
26: 118-127, 1951). 

Morphology: Two types of colonies are 
produced on agar: one small, flat, regular, 
white; the other thick, irregular, 
yellowish. Sporophores form spirals. Spores 
oval, 0.8 to 1.5 by 0.8 up. 


large, 


Sucrose nitrate agar: Growth limited. 
Aerial mycelium powdery, white. No soluble 


pigment. 

Nutrient agar: Growth poor, thin, yellow- 
ish. Aerial mycelium powdery, white. Solu- 
ble pigment brown. 

Peptone agar: Growth limited, cream- 
colored. Aerial mycelium powdery, white. 
Soluble pigment very slight, brown-reddish. 

Starch agar: Growth thin. Aerial myce- 
lum powdery, white. No soluble pigment. 

Potato: Punctiform colonies growing to- 
gether as thick crust, orange-reddish in 
color. Aerial mycelium limited, white, ap- 
pearing very slowly. No soluble pigment. 

Gelatin: Growth poor, flaky, white. 
Liquefaction limited. 

Milk: Growth Aerial 


At 25°C no coagulation; at 


mycelium 
Bye ©. 


coagulation after 20 days; no peptonization. 


slow. 
white. 


Nitrate reduction: Positive. 
Production of H.S: Negative. 
Source: Dog septicemia (thoracic, ab- 
dominal, and brain lesions). 

femarks: Said to be pathogenic for 
guinea pig and rabbit. Culture grown in the 


laboratory not pathogenic for dogs. 


97. Streptomyces gardnert (Waksman and 
Henrici) nov. comb. (Gardner, A. D. and 
Cham: i. Brit. J.. Exptl. Pathol: 232: 123. 
1942; Waksman, S. A., Horning, E. S. 
Welsch, M., and Woodruff, H. B. Soil Sci. 
04: 289, 1942). 

Morphology: When grown on oatmeal 
agar, aerial mycelium thin, largely at edge 
of growth, consisting of short, straight to 


216 


wavy sporophores, often produced in clus- 
ters; no spirals. 

Glucose-asparagine agar: Growth brown- 
ish, lichenoid, with wide cream-colored 
edge; reverse yellowish. Aerial mycelium 
white to grayish, gradually covering surface. 
No soluble pigment. 

Nutrient agar: Growth 
elevated, lichenoid, doughy consistency. No 
Soluble pigment faint 


cream-colored, 
aerial mycelium. 
brownish. 

Potato: Growth reddish- 
brown. No aerial mycelium. Pigment around 


barnacle-like, 


erowth grayish-brown. 

Gelatin: Surface ring cream-colored. Liq- 
uefaction medium. Soluble pigment deep 
brown, gradually diffusing through liquefied 
portion. 

Tryptone broth: Growth occurs in form 
of small pellets at the base of the flask; later, 
a thin surface pellicle is produced. Soluble 
pigment produced slowly, black. 

Temperature: Good growth at 25°C; slow 
growth at 37°C. 

Antagonistic properties: 
synthetic and organic media 


Produces on 
an antibiotic, 
proactinomyein, active against bacteria. 
Source: Isolated as an air contaminant. 
Remarks: This species was first classified 
as a Nocardia (Bergey’s Manual, 7th ed.). 
Recent evidence, comprising both cultural 
(Cummins and 


and chemical 


Harris, 1958), suggests its transfer to the 


properties 


genus Streptomyces. It is closely related to 
S. aureofaciens. 


Type culture: IMRU 3834. 


98. Streptomyces garyphalus Harris et al., 
1955 (Harris, D. A., Ruger, M., Reagan, 
M. A., Wolf, F. J., Peck, R. L., Wallick, H., 
and Woodruff, H. B. Antibiotics & Chemo- 
therapy 5: 183-190, 1955). 

Morphology: Sporophores straight, with- 
out spirals. Spores rod-shaped, 0.8 to 1.1 by 
Lat to: 129 iq: 

Growth colorless. 


Sucrose nitrate agar: 


THE ACTINOMYCETES, Vol. II 


Aerial mycelium grayish-white. No soluble 
pigment. 

Glucose-asparagine agar: Growth color- 
less. Aerial mycelium white. No soluble pig- 
ment. 

Modified glucose-asparagine agar: Growth 
powdery, pinkish-white, reverse buff. Aerial 
mycelium seashell-pink. No soluble pigment. 

Calcium malate agar: Growth colorless 
No aerial mycelium. 

Nutrient agar: Growth colorless. Aerial 
mycelhum grayish-white. Soluble pigment 
faint brown. 

Yeast extract-glucose agar: Growth excel- 
lent. Aerial myceluum = grayish-white, be- 
coming pinkish-gray and _ finally seashell- 
pink. Soluble pigment faint brown. 

Starch-tryptone agar: Growth good. Aerial 
mycelium gray. Soluble pigment dark brown. 

Peptone-glucose agar: Growth cream- 
colored. Aerial mycelium grayish-white, be- 
coming pink. Soluble pigment faint brown. 

Starch agar: Growth Aerial 
mycelium white to gray. Hydrolysis. Soluble 
pigment faint brown. 


excellent. 


Gelatin: Grayish-white ring and sub- 
merged pellicle. Pigmented layer dark 
brown; becomes greenish-brown when 


shaken. Medium liquefaction. 

Milk: Faint grayish-white tinge. Slow 
peptonization turning dark purple at first 
and later brownish-purple. Reaction acid, 
pH 6.4. 

Nitrate reduction: Strong. 

Potato: Growth heavy, wrinkled. Aerial 
mycelium grayish-black. Potato darkened. 

Cellulose: No decomposition. 

Antagonistic properties: Produces an anti- 
biotic, pb-4+-amino-3-isoxazolidone (novobio- 
cin). 

Habitat: Soil. 

99. Streptomyces  gedanensis — (Lohlein, 
1909) Miller, 1950 (Lohlein, M. Z. Hyg. 
Infektionskrankh. 63: 1-16, 1909; Miller, R. 
Medizinsche Mikrobiologie, 4th ed. 1950, 
Urban & Schwarzenberg, Miinich, p. 294). 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Morphology: Aerial mycelium consists of 
short, gnarled hyphae. Spores short, oval to 
spherical. 

Synthetic agar: Growth dark to almost 
black, with dark reverse. Aerial mycelium 
abundant, mouse-gray. No soluble pigment. 

Nutrient agar: Growth thin, colorless. No 
aerial mycelium. No soluble pigment. 

Glucose agar: Growth cream-colored, be- 
coming black with light margin. Aerial myce- 
lum abundant, mouse-gray. 

Potato: Growth lichenoid, cream-colored 
to brownish. No aerial mycelium. No soluble 
pigment. 

Gelatin: Growth thin, flaky. No soluble 
pigment. Rapid liquefaction. 

Milk: Surface 
peptonization. 

Starch media: Growth yellowish to cream- 
colored. Aerial mycelium light gray. Hy- 


ring cream-colored. No 


drolysis strong. 

Nitrate reduction: Negative. 

Production of HS: Negative. 

Source: Sputum of patient with chronic 
lung disease. 

Type culture: IMRU 3417. 

100. Streptomyces  gelaticus (Waksman, 
1919) Waksman and Henrici, 1948 (Waks- 
man, 8S. A. Soil Sci. 8: 165, 1919). 

Synonym: Streptomyces hepaticus. 

Morphology: Sporophores produce open 
spirals. According to Anderson ef al. (1956), 
the organism does not produce spirals. 

Sucrose nitrate agar: Growth colorless, 
spreading, chiefly deep into the medium. 
Aerial mycelium thin, white, turning grayish. 

Nutrient agar: Growth only on surface, 
wrinkled, cream-colored. 

Glucose agar: Growth abundant, spread- 
ing, white. 

Potato: Growth abundant, much wrin- 
kled, greenish, becoming black with yellow- 
ish margin. 

Gelatin: Produces flaky, cream-colored 
sediment. Good liquefaction. 


Pa ef 


Milk: 
peptonization. 

Starch: Growth thin, spreading, cream- 
colored. Hydrolysis. 

Nitrate reduction: Positive. 

Production of H.S: Negative. 

Temperature: Optimum 25°C. 


Pinkish ring. Coagulation and 


Antagonistic properties: Produces elaio- 
mycin. 

Habitat: Soil. 

temarks: Various. related 
been described by Gause ef al. (1957); these 
include A. 


forms have 
griseorubens, A. rubiginosus, and 
KrassilInikov (1959) 
siders this organism as belonging to the S. 


A. atroolivaceus. con- 


albus group. 
Type culture: IMRU 3323. 


101. Streptomyces glaucus (Lehmann and 
Schiitze emend. Krassilnikov, 1941) Waks- 
man (KrassiInikov, N. A. Actinomycetales. 
Izvest. Akad. Nauk. SSSR, Moskau, p. 46, 
1941). 

Morphology: Sporophores form compact 
spirals with 3 to 5 turns. Spores oval to 
spherical, 1.0 by 0.8 uy. 

Sucrose nitrate agar: Growth colorless: 
soluble pigment brown. Aerial mycelium at 
first white, then becoming bright green. 

Nutrient heavy. Aerial 
mycelium green. 

Potato: Growth heavy. Aerial mycelium 


Growth 


agar: 


velvety, green. 
Gelatin: Liquefaction slow. Melanin-nega- 
tive. 
Milk: 
coagulation by some strains. 
Starch: Hydrolysis rapid. 
Cellulose: Growth good. 
Nitrate reduction: Positive. 


Peptonization slow, with prior 


Sucrose: Poor inversion. 
Paraffin: Growth good. 
Antagonistic properties: All — strains 
strongly antagonistic. 

Habitat: Soil. 

temarks: Numerous cultures belonging 
to this organism or closely related to it have 


218 


been described under a variety of different 
names. It 1s sufficient to mention S. caelestis, 
which produces an antibiotic, celesticetin, 
described by DeBoer et al. (1954) and A. 
glaucescens, together with a variety badius, 
described by Gause et al. (1957). 


102. Streptomyces globisporus (kXrassilni- 
kov, 1941) Waksman (Krassilnikov, N. A. 
Actinomycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 48, 1941). 

Morphology: Sporophores 
wavy, often gathered in clusters or tufts; no 
spirals. Spores oval (1.2 to 1.4 by 1.8 to 
2.0 uw) or spherical (0.9 to 1.4 yu). 

Starch-KNO; agar: Growth abundant, 
colorless. Aerial mycelium light yellow to 
greenish-yellow with pinkish No 
soluble pigment. 

Glucose-peptone agar: Growth colorless 
or greenish. Aerial mycelium creamy, seldom 
greenish-yellow. Soluble pigment absent or 
faint yellowish. 

Gelatin: Rapid liquefaction. Soluble pig- 
ment absent or hight brownish. 

Potato: Growth colorless or 
Aerial mycelium 
brownish or colorless. 

Milk: No coagulation; rapid peptoniza- 
tion. 

Starch: Weak hydrolysis. 

Invertase: None. 

Nitrate: Reduced to nitrite. 

Cellulose: No or poor growth. 
Some 


straight or 


tinge. 


brownish. 


greenish-yellow. Plug 


Antagonistic properties: straims 
suppress gram-positive bacteria. 

Habitat: Soil. 

temarks: Krassilnikov recognized several 
substrains of the species on the basis of 
milk coagulation, proteolysis, and pigmenta- 
tion of aerial mycelium. It is sufficient to 
mention A. globisporus vulgaris, A. globis- 
porus griseus, A. globisporus lactis, A. globis- 
porus diastaticus, A. globisporus flaveolus, A. 
globisporus circulatus, A. globisporus scabies, 


and A. globisporus albus. This heterogeneous 


THE ACTINOMYCRETES, Vol. II 


collection is most unfortunate, since these 


ce 9) 


species”? show differences in color of aerial 
mycelium, in formation of soluble pigments, 
etc. 

Krassilnikov (1949) considered the strep- 
tomycin-producing organism as a_ variety 
of this species, designating it at first as A. 
globisporus streptomycini, and later as A. 
streptomycin, A. Krainsky 
distinguished from this species on the basis 
of the fact that the sporophores of the latter 
exhibited spiral formation. This again was 
the cause of much confusion in nomenclature 


griseus was 


of the streptomycin-producing organism in 
the literature of the Soviet Union. 

Later, Krassilnikov (1958) divided the A. 
globisporus group, on the basis of antago- 
nistic effects, Into a number of subgroups, 
including A. vulgaris, A. toxicus, A. levoris, 
A. bacillaris, A. fluorescens, A. raffinosus, A. 
longisporus, and A. grisinus. 

Gause et al. (1957) described A. 
porus in the series “‘helvolus,’’ comprising 


globis- 


the S. griseus group; they also listed several 
additional forms belonging to S. globisporus 
under the names A. caucasicus and A. 
cyanofuscatus. The above description is 
based upon the comparison made by Gause 
et al. of six cultures and Krassilnikov’s 
authentic strain. 


103. Streptomyces globosus (Xrassilnikov, 
1941) Waksman (Krassilnikov, N. A. Actino- 
mycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 58, 1941). 

Morphology: Sporophores straight, short, 
slightly branched, wavy. Spores spherical to 
oval. 

Agar media: Substrate growth brown to 
dark brown. Aerial mycelium dark gray, 
velvety. Soluble pigment dark brown. 

Gelatin: Weak liquefaction. No soluble 
pigment. Melanin formation questioned by 
Hoffmann (1958). 

Potato: Soluble pigment red-brown (Hoff- 
mann, 1958). 


DESCRIPTION OF 


Milk: Questionable 
peptonization. 

Starch: Hydrolysis. 

Cellulose: ¢ 
No inversion. 
: Positive. 


coagulation; good 


wood growth. 
Sucrose: 
Production of H.S 
Antagonistic properties: No activity. 
Habitat: Soil, food products, potatoes. 
Type culture: IMRU 3736 


104. Streptomyces gougeroti (Duché, 1934) 
Waksman and Henrici, 1948 (Duché, J. Les 
actinomyces du groupe albus. P. Lechevalier, 
Paris, 1934) 

Morphology: Aerial hyphae short, gnarled. 
Spores oval. 

Glucose slow as 
punctiform with 
smooth edge. No aerial mycelium. No soluble 


agar: Growth 
cream-colored 


nitrate 
colonies; 


pigment. 

Glucose-asparagine agar: Growth colorless 
to yellowish. Aerial mycelium thin, white. 
No soluble pigment. 

Nutrient agar: Growth »am-colored 
with brownish reverse. Aerial mycelium 
thin, white. Soluble pigment faint yellowish. 


Potato: Growth slow, greenish tinged. 
Aerial mycelium thin, white. No soluble pig- 
ment. 

Gelatin: Surface growth heavy, cream- 


colored. Aerial mycelium thin, white. Lique- 
faction strong. Melanin-negative. 
Milk: Growth cream-colored. 
celium thin, white. Peptonization rapid. 
Coagulated serum: Growth cream-colored. 
Aerial mycelium white. Liquefaction rapid. 
Starch: Hydrolysis rapid. 
Nitrate reduction: Negative. 
Production of H.S: Negative. 
properties: Active 


Aerial my- 


Antagonistic against 
fungi. 
Remarks: 
intermediate 
abundant aerial mycelium, and S. almquist?, 
with its very scant aerial mycelium. 
Type culture: IMRU 3590. 


This culture is believed to be 


between S. albus, with its 


SPECIES OF 


STREPTOMYCES 219 
105. Streptomyces gracilis Eee and 
Burr, 1926) Waksman (Millard, W. A. and 


Burr, S. Ann. Appl Biol. 13: 580, ae 

Morphology: 
Spores oval or spherical, 0.8 to 0.9 by 0.8 yp. 
fern-like, 
gray to 


Sporophores form. spirals. 
nitrate Growth 
pale gray. Aerial mycelium scant, 
buff. Soluble pigment cream-colored. 
Nutrient potato agar: 
buff to dark brown or almost black. Aerial 


Sucrose agar: 


Growth vinaceous- 


mycehum gray. Soluble pigment light golden 
brown. 

Potato: 
gray to buff 

Gelatin: 
white. Liquefaction 
pink to dark golden brown. 

Milk: Surface growth good. Aerial myce- 
lium white in the form of a ring and specks 
Coagulation slow, followed by 


Aerial mycelium abundant, olive- 
f. Plug pigmented light brown. 

Growth gray. Aerial mycelium 

rapid. Soluble pigment 


on surface. 
rapid peptonization. 
Starch: Positive hydrolysis. 
Nitrate reduction: Positive. 
Tyrosinase reaction: Negative. 
Temperature: Grows well at 37.5° 
Habitat: 


106. Streptomyces 


Potato scab. 


grisenus  Waksman 
(Reynolds, D. M. and Waksman, S. A. 
J. Bacteriol., 55: 739-751, 1948; Okami, Y. 
J. Antibiotics (Japan) 3: 95-97, 1950). 
Morphology: Straight sporophores  pro- 
duced clusters or tufts, without spirals. 
Spores rod-shaped, 1.0 to 1.8 by 0.8 to 1.0 xu. 
nitrate agar: Substrate growth 
-am-colored to brownish. 


Sucrose 
wrinkled, reverse cr 
Aerial mycelium white to 
with light greenish tinge (lesser tendency to 


cream-colored 


grass-green coloration, more of a cream- 
color). No soluble pigment. 
Starch agar: Colorless 


Aerial mycelium grayish-olive. 


to cream-colored 
growth. Hy- 
drolysis rapid. 

Potato: Growth wrinkled, yellowish-white. 
Aerial with olive 


tinge. 


mycelium grayish-white 


220 


Gelatin: Growth cream-colored with 
brownish tinge. Aerial mycelium absent, or 
scant, white. Liquefaction rapid. 

Milk: Growth cream-colored. Coagulation 
and peptonization. 

Tyrosine agar: No pigment produced. 

Nitrate reduction: Positive. 

Production of HS: Negative. 

Carbon utilization: Okami (1950) re- 
ported that the grisein-producing organism 
GS. griseinus) grows more readily in synthetic 
media contaming and 
sucrose than the streptomycin-producing S. 
griseus. According to Benedict et al. (1955), 
the former utilizes xylose, L-arabinose, and 


glucose, glycerol, 


rhamnose, but S. griseus utilizes only xylose. 

Phage sensitivity: Not sensitive to phages 
effective against S. griseus strains. 

Pigments: No soluble pigments on calcium 
malate or succinate media, whereas S. gr7- 
seus forms green and yellow pigments on 
these media, according to Benedict and Lind- 
enfelser (1951). 

Antagonistic properties: Produces the an- 
tibiotic grisein. Albomycin, produced by A. 
subtropicus (Gause, 1955), is an identical or 
closely related compound (Waksman, 1957; 
Thrum, 1957). 

femarks: S. griseus and S. griseinus show 
other striking differences. There are some 
close resemblances between these and the 
viomycin-producing cultures. Thus, on tyro- 
sine-starch agar, certain S. griseus strains 
form a dark pigment in the agar, whereas S. 
griseinus strains resemble the viomycin-pro- 
ducing cultures by not forming this pigment. 
S. griseinus and the viomycin group grow 
well on NaNO;, but S. griseus utilizes this 
compound poorly. A. subtropicus, described 
by Jkudrina and Kochetkova (1958), is 
closely related to, if not identical with SS. 
Griseinus. 

Type culture: IMRU 3478. 

107. Streptomyces griseobrunneus Waks- 
man, 1919 (Waksman, 8. A. Soil Sei. 8: 125 
127-1919): 


THE ACTINOMYCETES, Vol. II 


Morphology: Sporophores usually straight 
on most media; often a few short, open 
spirals are formed; tufts are produced on 
certain media. Spores oval-shaped. 

Sucrose nitrate agar: Growth cream-col- 
ored to yellowish-brown. Aerial mycelium 
appears early; powdery, olive-buff to water- 
green. No soluble pigment. 
malate Growth 
cream-colored. Aerial mycelium water-green 
in color. 


Glycerol-caletum 


agar: 


Glucose-asparagine agar: Growth yellow- 
ish-brown. Aerial mycelium pale olive-buff. 
No soluble pigment. 

Nutrient agar: Growth cream-colored, be- 
coming brown. Aerial mycelium abundant, 
white. Soluble pigment brown. 

Starch agar: Growth cream-colored to yel- 
lowish. Aerial mycelium white. Good hydrol- 
ysis. 


Ege media: Growth cream-colored with 


455 
brownish tinge. Aerial mycelium olive-buff. 
Soluble pigment purple. 

Potato: Growth brownish. Aerial myce- 
lium white, turning olive-buff. Soluble pig- 
ment faintly brown. 

Gelatin: Growth cream-colored, turning 
brown. Aerial mycelium white. Soluble pig- 
ment deep brown. Medium liquefaction. 

Milk: No coagulation; rapid peptoniza- 
tion. 

Nitrate reduction: Positive. 

Sucrose: No inversion. 

Cellulose: Good growth. 

Habitat: Sewage. 

Yemarks: This organism had been de- 
scribed by Waksman (1919) as Actinomyces 
218, but never named before. It was said to 
be closely related to S. griseus, differing from 
it by lesser proteolysis and production of a 
brown pigment on protein media. 

Type culture: IMRU 3068. 

108. Streptomyces griseocarneus Benedict 
et al., 1951 (Benedict, R. G., Lindenfelser, 
L. A., Stodola, F. H., and Traufler, D. H. 


J. Bacteriol. 62: 487-497, 1951; see also 


DESCRIPTION OF SPECIES OF STREPTOMYCES 221 


Grundy, W. E. Antibiotics & Chemother- 
apy 1: 309-317, 1951). 

Morphology: Sporulation occurs best on a 
carbon-free salt agar, to which 0.5 per cent 
soluble starch has been added. Sporophores 
straight, forming no spirals. Spores coccoid 
toroveal lletoslG2by O27 told.L a: 

Agar media: Aerial mycelium on some 
media powdery, becoming gray, but no spor- 
ulation. When sporulation occurs the myce- 
lium becomes light pink. 
limited, 


nitrate Growth 


white. Aerial mycelium white, no sporula- 


Sucrose agar: 
tion. 

Glucose-asparagine agar: Growth moder- 
ate. Aerial mycelium powdery, white, no 
sporulation. 

Caletum malate agar: Growth moderate, 
white. Aerial mycelium white, no sporula- 
tion. 

Nutrient agar: Growth moderate, cream- 
colored. No aerial mycelium. Soluble pig- 
ment light yellow-brown. 

Oatmeal agar: Growth luxuriant, brown. 
Aerial mycelium abundant, fluffy, white; no 
sporulation. No soluble pigment. 

Potato: Growth cream-colored. Aerial my- 
celium gray. Soluble pigment hght brown, 
turning dark brown. 

Gelatin: Growth cream-colored to brown. 
Rapid liquefaction. Soluble pigment dark 
brown. 

Milk: Growth dark brown to black. No 
coagulation; rapid peptonization. Soluble 
pigment brown. 

Starch: Hydrolysis. 

Carbon — utilization: 
starch, glycerol, calcium malate, and sodium 


Glucose, dextrin, 
succinate rapidly utilized. Mannose, mal- 
tose, inositol, and sodium acetate utilized 
slowly. Xylose, galactose, sorbose, sucrose, 
cellobiose, melibiose, lactose, mannitol, sor- 
bitol, sodium citrate, and potassium sodium 
tartrate not utilized. 

Nitrate reduction: Negative. 

Production of HS: Positive. 


Antagonistic properties: Produces hy- 
droxystreptomycin. 

Type culture: IMRU 3557; ATCC 12,628. 

109. Streptomyces griseochromogenes Yuku- 
naga et al., 1955 (Fukunaga, K., Misato, T. 
Ishu, I., and Asakawa, M. Bull. Agr. Chem 
Soc. Japan 19: 181-188, 1955). 

Morphology: Sporophores form closed spi- 
rals on starch agar; there are no spirals, or 
only curling tips, formed on sucrose nitrate 
and glucose-asparagine agars. Spores spheri- 
cal or oval, about 1.0 to 1.5 uw. 

Sucrose nitrate agar: Growth spreading, 
orange-cinnamon. Aerial mycelium white or 
light neutral gray. No soluble pigment. 

Glucose-asparagine agar: Growth — re- 
stricted, ivory-yellow, penetrating into the 
medium. No aerial mycelium, later white. 
No soluble pigment. 

Nutrient agar: Growth restricted, opales- 
cent. No aerial mycelium. Soluble pigment 
brown. 

Potato: Growth 
snuff-brown. Aerial mycelium 
mouse-gray. Color of plug dark brown to 
black around growth. 

Gelatin: Growth wrinkled, yellowish in 


wrinkled, 
white to 


abundant, 


liquefied portion. Aerial mycelium white, 
scant. Liquefaction medium. Soluble pig- 
ment dark brown to black. 

Milk: Growth as surface ring, brown. No 
coagulation; peptonization begins in 8 days 
at 37°C; not completed in 21 days. 

Nitrate reduction: Positive. 

Cellulose: No growth. 

Tyrosine agar: Growth orange-colored. No 
soluble pigment. 

Invertase: Positive. 

Carbon utilization: Glucose, p-fructose, 
p-galactose, maltose, lactose, raffinose, pb- 
mannitol, utilized. 
inulin, p-sorbitol, dulcitol, salicin, sodium 


DL-Inositol Rhamnose, 
acetate, sodium citrate, sodium succinate not 
utilized. 

Antagonistic properties: Produces blasti- 
cidins A, B, and C, active against fungi. 


temarks: S. griseochromogenes belongs to 
the group of chromogenic actinomycetes. S. 
resistomycificus differs from S. griseochromo- 
genes in the color of its aerial mycelium ob- 
served on various media, and also in that it 
produces an aerial mycelum on nutrient 
agar and a soluble pigment in glucose-aspara- 
gine agar. S. mirabilis has a different form 
of aerial mycelium and a different optimum 
temperature. S. flavochromogenes produces 
an aerial mycelium on nutrient agar and a 
grayish soluble pigment. S. olivochromogenes 
assumes a dark brown or black color of 
growth and shows an alkaline reaction in 
milk medium. S. dzastatochromogenes is quite 
similar to S. griseochromogenes in the ap- 
pearance of its growth on several media, but 
differs from it by producing a white or gray 
aerial mycelium on nutrient agar, and also 
by producing tyrosinase. 


110. Streptomyces griseoflavus (srainsky, 
1914) Waksman and Henrici, 1948 (Krain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
II, 41: 684, 1914). 

Morphology: Sporophores straight, mono- 
podially branched; no curvatures and no 
spirals produced. Spores oblong, 1.0 to 1.2 u, 
covered with short spines. (According to 
Ettlnger et al. (1958), open, regular spirals 
are formed.) 

Sucrose nitrate agar: 
brown to orange. Aerial mycelium gray to 


Growth reddish- 
yellowish-gray. Faint greenish-yellow soluble 
pigment. 

Glucose-asparagine agar: Growth citron- 
yellow. Aerial mycelium powdery, greenish- 
yellow changing to gray (Hoffmann, 1958). 

Calcium malate agar: Growth yvellowish- 
green-gray. 

Nutrient Growth 
covered with white to gray aerial mycelium. 


agar: cream-colored, 


Soluble pigment absent or, according to 
Hoffmann (1958), greenish-gray. 

Starch 
brownish center. Aerial mycelium absent or 


agar: Growth cream-colored with 


powdery, gray. Hydrolysis limited. 


THE ACTINOMYCETES, Vol. II 


Potato: Growth lichenoid, yellowish. Ae- 
rial mycelium powdery, white to gray. Mela- 
nin-negative. 

Gelatin: Growth cream-colored to brown- 
ish, covered with white to yellowish-gray 
aerial mycelium. Positive liquefaction. Mela- 
nin-negative. 

Milk: Growth cream-colored to yellowish; 
aerial mycelium thin, white. No coagulation; 
rapid peptonization. 

Cellulose: Growth good. Greenish-yellow 
soluble pigment, according to Hoffmann 
(1958). 

Nitrate: Strong reduction to nitrite. 

Production of HS: Negative. 

Invertase: Negative. 

Antagonistic properties: According to 
Waga (1953), a member of this group pro- 
duced an antibiotic, griseoflavin; this anti- 
biotic was later (Xuroya et al., 1958) found 
to be identical with novobiocin. Another 
antibiotic, grisamine, has also been reported. 

Remarks: According to Jensen (1930), the 
species is characterized by the grayish-yellow 
color of its aerial mycelium, which never 
assumes the distinct green shade of S. griseus. 
A detailed study of the life cycle of this 
organism has been made by Saito and Ikeda 
(1958). They found that between the pri- 
mary (vegetative phase) and the secondary 
(sporulation phase) mycelium, there may be 


‘ 


a transitional stage which comprises ‘‘nests,”’ 
“swollen bodies,” and ‘“‘clubs,’”’ correspond- 
ing to the “initial cells’’ reported in the litera- 
ture. 

111. Streptomyces griseolus (Waksman, 
1923) Waksman and Henrici, 1948 (Waks- 
man, 8. A. Actinomyces 96. Soil Sci. 8: 121, 
1919). 

Morphology: Sporophores short, straight, 
without spirals, some curling found on side 
branches. Spores spherical to oval-shaped to 
cylindrical. 

Growth colorless, 
thin, spreading, chiefly in the medium. 
Aerial mycelium at first gray, later becoming 


Sucrose nitrate 


agar: 


DESCRIPTION OF SPECIES OF STREPTOMYCES 223 


pallid neutral gray, with yellowish tone. 
Faint brownish soluble pigment. 

Malate-glycerol agar: Growth brownish. 
Aerial mycelium lght mouse-gray. Soluble 
pigment faint brownish. 

Glucose-asparagine agar: Growth cream- 
colored, turning dark. Aerial mycelium deep 
dull gray. No soluble pigment. 

Nutrient agar: Brownish growth, with 
smooth surface. Aerial mycelium white with 
gray tinge. Soluble pigment brown. Mela- 
nin-negative. 

Potato: Growth cream-colored, becoming 
black. Aerial mycelium white with greenish 
tinge. Soluble pigment brown to black. 

Gelatin: Yellowish flaky pellicle and sedi- 
ment. Aerial mycelium white. Gradual lique- 
faction. Faint browning of medium. 

Milk: Growth abundant, pink pellicle. 
Slow coagulation and good peptonization. 

Starch media: Growth grayish-brown with 
dark ring. Aerial mycelium gray. Slight hy- 
drolysis of starch. 

Cellulose: Scant growth. 

Invertase: Negative. 

Nitrate reduction: Positive. 

Production of H.S: Negative. 

Temperature: Optimum 25°C. 

Antagonistic properties: Some strains 
show considerable activity against various 
bacteria. Several antibiotics (phagomycin, 
fermicidin, anisomycin, oxytetracycline, gris- 
eomycin) were isolated from cultures de- 
scribed as strains of S. griseolus. 

Habitat: Soil. 

Remarks: Ettlinger et al. (1958) considered 
this organism as related to S. olivaceus. 
Krassilnikov (1949) considered it as a strain 
of A. candidus. Hoffmann (1958) isolated a 
strain of this species from potato scab. Al- 
though this organism is usually described as 
melanin-negative, Krassilnikov (1941) and 
Hoffmann consider it as melanin-positive. 

Type culture: IMRU 3325. 


112. Streptomyces griseoluteus 
i 
1951 (Umezawa, H., Hayano, 5., 


Umezawa 


et al., 


Maeda, K., Ogata, Y., and Okami, Y. J. 
Antibiotics (Japan) 4: 34-40, 1951; Okami, 
Y. zbid. 5: 477-480, 1952). 

Morphology: 
podial and irregular branching, flexible and 
hooked. Spores oval to cylindrical, 1.0 to 1.2 
by 1.8 to 2.2 yu. 

Sucrose nitrate agar: Growth thin, color- 


Sporophores with mono- 


less to cream-colored. Margin plumose, pene- 
trating into medium. Aerial mycelium pow- 
dery, grayish-white to light drab. Soluble 
pigment absent or yellowish-brown. 

Glucose-asparagine agar: Growth § wrin- 
kled, cream-colored. Aerial mycelium. thin, 
white. Pigment reddish-brown. 

Nutrient agar: Growth wrinkled, trans- 
parent. Aerial mycelium thin, white, pow- 
dery. Soluble pigment absent or yellowish- 
brown. 

Potato: Growth 
cream-colored. Aerial mycelium dusty white, 
thin. Plug becoming slightly brownish. 

Gelatin: No growth. 

Milk: Surface ring cream-colored. Aerial 


abundant, wrinkled, 


mycelium in form of white patches. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Production of H.S: Negative. 

Antagonistic properties: Produces griseo- 
lutein. 

Type culture: IMRU 3674; 3729. 

115. Streptomyces griseoplanus Backus et 
al., 1957 (Backus, E. J., Tresner, H. D., and 
Campbell, T. H. Antibiotics & Chemother- 
apy 7: 532-541, 1957). 

Morphology: Sporophores arise as tangled 
and curved and often loosely spiraled chains 
of spores. Spores globose to elliptical, 0.6 to 
2 to 1.5 u (Fig. 40). 
Growth 


2 DY 


Sucrose nitrate agar: colorless. 
Aerial mycelium scant, white to gray. 
Glucose-asparagine agar: Growth gray to 
light pinkish. No aerial mycelium. 
Nutrient agar: Growth ivory-yellow. No 


aerial mycelium. Melanin-negative. 


224 THE ACTINOMYCETES, Vol. II 


FragureE 40. Sporophores of S. griseoplanus (Reproduced from: Backus, E. J. 


Chemotherapy 7: 537, 1957). 


Starch agar: Growth colorless to yellow- 
ish. Aerial mycelium scant, white. 

Potato plug: Growth light brownish. Ae- 
rial mycelium scant, white to gray. Apex of 
plug browned. 

Milk: No growth. 

Cellulose: No growth. 

Production of HoS: Negative. 

Carbon utilization: Extremely limited. 
with ammonium sulfate as source of nitro- 
gen. With aspartic acid, organism utilizes 
L-arabinose, D-xylose, and glucose. Fair to 
moderate growth on lactose, b-raffinose, pb- 
trehalose, and salicin. Sucrose, p-fructose, 


pb-mannitol, 7-inositol, adonitol, p-melezitose, 


i. 


et al. Antibiotics & 


L-rhamnose, esculin,p-melibiose, and dextrin 
utilized poorly or not at all. 

Antagonistic properties: Produces anti- 
biotic alazopeptin. 

Habitat: Grassland soil. 

femarks: This organism is closely related 
to S. flavogriseus. 


114. Streptomyces griseoruber Yamaguchi 
and Saburi, 1955 (Yamaguchi, T. and Sa- 
buri, Y. J. Gen. Appl. Microbiol. 1: 201-235, 
1955). 

Morphology: Aerial hyphae produce open 
and closed spirals on sucrose nitrate and on 
starch agars. Spores short, cylindrical, 0.5 to 
0.9 by 0.9 to 1.2 p. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 225 


nitrate agar: Growth reddish- 


orange when freshly isolated, but changes to 


Sucrose 
colorless or whitish on repeated transfer. 
Aerial mycelium powdery, drab-gray. No 
soluble pigment. 

Calcium malate agar: 
glossy, pinkish-gray, later becoming dull 
purplish. Aerial mycelium powdery, olive- 
gray. Soluble pigment absent or faint yel- 


Growth at first 


lowish-brown. 

Nutrient agar: Growth at first light olive- 
gray, later becoming brown. Aerial myce- 
lum absent or scanty, white. Soluble pig- 
ment brown. 

Starch agar: Growth light reddish-orange 
to reddish-purple. Aerial mycelium powdery, 
olive-gray. Soluble pigment faint yellow to 
faint yvellowish-pink. Strong hydrolysis. 

Potato: Growth wrinkled, at first olive- 
gray to dark yellowish-brown, later becom- 
ing dark reddish or black. Aerial mycelium 
absent or scanty, white. Soluble pigment 
deep purple to black. 

Gelatin: Growth light yellowish-brown on 
surface. No aerial mycelium. Soluble pig- 
ment brown to lght yellowish-green. Lique- 
faction weak to medium. 

Milk: Growth deep brown at 37°C, but 
cream-colored to light yellowish-brown, and 
occasionally with pinkish tone at 25°C. Sol- 
uble pigment grayish-yellow-brown at 37°C, 
but sometimes faint yellowish-brown with 
pinkish shade at 25°C. Coagulation begins in 
3 days, followed by peptonization. 

Cellulose: No growth. 

Carbon utilization: Utilizes p-xylose, L- 
arabinose, L-rhamnose, lactose, inositol, sali- 
cin, sodium acetate; does not utilize sucrose, 
raffinose, inulin, mannitol, sorbitol, citrate, 
and succinate. 

Antagonistic properties: Active 
gram-positive and acid-fast bacteria; pos- 


against 


sesses antitrichomonal activity. 
telated to the S. 
and to S. erythrochromogenes. 


temarks: ruber group 


115. Streptomyces griseoviridis Anderson 


et al., 1956 (Anderson, L. E., Ehrlich, J., 
Sun, 8. H., and Burkholder, P. R. Antibiot- 
ics & Chemotherapy 6: 100-115, 1956). 

Not S. griseoviridus. 

Morphology: Sporophores straight or 
curved, with open and closed spirals on 
lateral branches. Spores spherical to ovoid, 
0.6 to 1.5 by 0.8 to 2.1 yu. 

Starch-ammonium sulfate agar: Growth 
tan-gray to black. Aerial mycelium tan to 
light brown. 

Glycerol-asparagine agar: Growth light 
vellow to gray. Aerial mycelium pink-tan to 
eray-green. 

Calcium malate agar: Growth yellow-tan 
to gray. Aerial mycelium light brown. Slight 
hydrolysis of starch. 

Nutrient 
ereen-gray to brown. Aerial mycelium light 


agar: Growth yellow-tan to 
gray-pink to light gray-green. Soluble pig- 
ment light brown. 

Glucose-tryptone light 
brown to red-brown to dark brown or black. 


agar: Growth 
Aerial mycelium pink-gray to light brown 
to green-brown. Soluble pigment brown; red- 
brown near growth. 

Gelatin: Fairly rapid liquefaction. Soluble 
pigment light brown to dark brown. 

Milk: Peptonization. 

Starch: Hydrolysis. 

Carbon utilization: 
cellobiose, dextrin, galactose, glucose, glyc- 
mannitol, 


Utilizes arabinose, 


erol, lactose, levulose, maltose, 
mannose, rhamnose, starch, trehalose, and 
xylose. Does not utilize esculin, adonitol, 
dulcitol, z-inositol, inulin, melezitose, meli- 
biose, raffinose, salicin, sorbitol, and sucrose. 

Antagonistic properties: A source of gris- 
eoviridin and viridogrisein (etamycin). 

Habitat: Texas soil. 

Type culture: IMRU 3735. 

116. Streptomyces griseus Waksman and 
Henrici, 1948 (Waksman and Henrici, Ber- 
gey’s Manual, 6th ed. 1948, p. 948; Waks- 
man, §. A. and Curtis, R. E. Soil Sei. 1: 


119-120, 1916; Waksman, 8. A., Reilly, 


DIG 


Ei Cs"and Harris: DA. Je Bacteriol. 56: 
259, 1948; Waksman, S. A. Proc. Natl. 
Acad. Sei. U.S. 45: 1043-1047, 1959). 

Synonyms: Actinomyces globisporus Wras- 
silnikov, 1941. Actinomyces globisporus sub- 
sp. streptomycinta (Waksman) Irassilnikov, 
1949. Actinomyces streptomycini Wrassilni- 
kov, 1957. 

Morphology: Sporophores straight, pro- 
duced in tufts (Fig. 41). Spores spherical to 
oval, 0.8 by 0.8 to 1.7 uw; surface smooth (PI. 
eben). 

Sucrose nitrate agar: Growth thin, spread- 
ing, colorless, becoming olive-buff. Aerial 
mycelium thick, powdery, water-green. Pig- 
ment insoluble. 

Nutrient agar: Growth abundant, almost 
transparent, cream-colored. Aerial mycelium 
powdery, white to light gray. No soluble 
pigment. 

Glucose agar: Growth elevated in center, 
radiate, cream-colored to orange, erose mar- 


gin. 


Substrate and aerial mycelium of 


FIGURE 41. 


S. griseus. 


THE ACTINOMYCETES, Vol. II 


Starch media: Growth thin, spreading, 
transparent. Hydrolysis strong. 

Tyrosine agar: Dark pigment often pro- 
duced. 

Potato: Growth wrinkled, yellowish to 
brownish, covered with white, powdery aer- 
ial mycelium. 

Gelatin: Greenish-yellow or cream-colored 
surface growth with brownish tinge. Rapid 
liquefaction. 

Milk: Cream-colored ring; 
with rapid peptonization, becoming alkaline. 

Cellulose: Scant to fair growth. 

Nitrate reduction: Positive. 

Pigments: Produces green or yellow solu- 
ble pigment on calcium malate and succinate 
media. 

Production of HS: Negative. 

Carbon sources: See S. griseinus. 

Antagonistic properties: Strongly antago- 
nistic. Produces antibiotic streptomycin, ac- 
tive against a large number of bacteria and 
actinomycetes, but not against most fungi or 
viruses; also produces cycloheximide, active 
upon fungi. Resistant to streptomycin-pro- 
ducing organisms and to streptomycin. 

Remarks: An extensive literature has ac- 
cumulated on the nature of this organism 
(KXoreniako and Nikitina, 1959), on its phage 
sensitivity (xoerber et al., 1950), antibiotic 
production (Waksman, 1949), ete. 

Habitat: Soils, river muds, 
chicken. 

Type culture: IMRU 3463. 


coagulation 


throat of 


117. Streptomyces hachijoensis Yamaguchi, 
1954 (Yamaguchi, T. J. Antibiotics (Japan) 
7A: 10-14, 1954). 

Morphology: Aerial hyphae short, straight, 
0.6 to 1.2 uw. Secondary verticils produced. 
Spores cylindrical, 0.8 to 1.0 by 1.5 to 1.8 yu. 

Sucrose nitrate agar: Growth restricted, 
colorless; reverse yellowish. Aerial mycelium 
white, changing to pinkish-buff. No soluble 
pigment. 

Calcium malate agar: Growth colorless to 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


yellow. Aerial mycelium white to pale pink- 
ish-buff. Soluble pigment absent. 

Nutrient agar: Growth cream-colored, 
wrinkled. Aerial mycelium powdery, shade of 
pale ochraceous-buff. No soluble pigment. 

Potato: Growth cream-colored to yellow- 
ish, wrinkled, raised. Aerial mycelium white. 
Soluble pigment around growth faint pur- 
plish. 

Blood agar: Growth yellow to brownish- 
yellow. Aerial mycelium flocculent, white. 
Soluble pigment dark. Positive hemolysis. 

Gelatin: Growth yellow to brown. No 
aerial mycelium. Rapid liquefaction. 

Milk: Surface ring yellow to brown. Aerial 
mycelium in form of white patches. Soluble 
pigment pinkish to orange. Coagulation fol- 
lowed by peptonization. 

Nitrate reduction: Negative. 

Cellulose: No growth. 

Antagonistic properties: Produces an anti- 
fungal agent, trichomycin, a member of the 
candicidin group of antibiotics. 

Remarks: Resembles S. rubrireticult. Bli- 
nov (1958) described a variety (fuscatus) of 
this species, as a producer of candicidin- 
type antibiotics. 


118. Streptomyces halstedii (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 
(Waksman, S. A. and Curtis, R. E. Soil Sci. 
J: 124, 1916; 8: 121, 1919). 

Morphology: Sporophores 
spirals. Spores oval or rod-shaped, 1.0. to 
eZ 1.2 tOM Su: 

Sucrose nitrate agar: Substrate growth 


form closed 


abundant, spreading, raised, at first light 
colored, becoming dark to almost black. 
Aerial mycelium white, turning dull gray. 
No soluble pigment. 

Glycerol malate agar: Growth dark. Aerial 
mycelium deep mouse-gray. 

Nutrient agar: Growth restricted, wrin- 
kled, cream-colored. No aerial mycelium. 
Melanin-negative. 


Glucose-asparagine agar: Growth  wrin- 


227 


kled, center elevated, edge lichenoid, color- 
less, becoming brown. No aerial mycelium. 

Potato: Growth abundant, moist, wrin- 
kled, cream-colored with green tinge. 

Gelatin: Small, cream-colored masses of 
growth in bottom of tube. Rapid liquefae- 
tion. No soluble pigment. 

Milk: 
and slow peptonization. 

Starch media: Growth abundant, glossy, 
brownish. No aerial mycelium. Rapid hy- 


Cream-colored ring. Coagulation 


drolysis. 

Cellulose: No growth. 

Nitrate: Reduction to nitrite. 

Production of H»S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Strongly antag- 
onistic; some strains show only antifungal 
activity; some strains produce carbomycin. 

Habitat: Soil. 

Remarks: Several closely related forms 
have been described. According to Ettlinger 
et al. (1958), the strains examined produce 
no spirals and belong to S. oldvaceus. Ac- 
cording to Okami and Suzuki (1958), the 
sporophores are wavy, seldom forming hooks 
or primitive spirals. Gause et al. (1957) de- 
scribed a closely related form as A. griseoin- 
carnatus. 


Type culture: IMRU 3328. 


119. Streptomyces hawaiiensis Cron et al., 
1956 (Cron, M. J., Whitehead, D. I°., Hooper, 
I. R., Heinemann, B., and Lein, J. Anti- 
biotics & Chemotherapy 6: 63-67, 1956). 

Morphology: Sporophores produce spirals 
on some media. Spores oval, 0.6 to 0.8 by 
O57 to des we 

Sucrose nitrate agar: Growth faint yellow. 
Aerial mycelium sparse, white to flesh-col- 
ored. Soluble pigment faint tan or absent. 

Glucose-asparagine agar: Growth light 
brownish. Aerial mycelium moderate, white 
to gray. Soluble pigment faint tan or absent. 

Nutrient agar: Growth gray with light 
Aerial mycelium limited, 


brown reverse. 


223 
wood-ash to steel-gray. Soluble pigment 
brown. 

Potato: Growth gray. Aerial mycelium 
limited, gray. Soluble pigment dark brown, 
almost black. 

Milk: No coagulation or peptonization. 
Slight acid reaction. Soluble pigment green- 
ish-brown. 

Gelatin: Slight liquefaction at 26°C in 19 
days. Soluble pigment brown. 

Starch: Shght hydrolysis in 7 days at 28- 
30°C. 

Blood agar: No hemolysis. Soluble pig- 
ment black. 

Production of H.S: Positive. 

Carbon Utilizes 
rhamnose, glucose, galactose, fructose, su- 


utilization: arabinose, 
crose, maltose, lactose, xylose, raffinose, cell- 
obiose, dextrin, inulin, soluble starch, glyc- 
erol, inositol, mannitol, sodium acetate, 
sodium citrate, and calcium malate. Does 
not utilize sorbitol, dulcitol, sodium oxalate, 
sodium salicylate, sodium tartrate, and so- 
dium succinate. 

Antagonistic properties: Produces a poly- 
peptide antibiotic, bryamycin. 

Remarks: S. hawaziensis is a chromogenic 
form which produces a soluble, dark brown 
pigment on protein media and a white to 
eray aerial mycelium. The organism is fur- 
ther characterized by spiral formation in the 
aerial mycelium and weak proteolytic ac- 
tivity in gelatin and milk. 

Streptomyces phaeofaciens possesses cul- 
tural and morphological characteristics simi- 
lar to those of S. hawaziensis, but differs in 
its rapid peptonization of milk and produc- 
tion of an antifungal substance inactive on 
bacteria. 

S. hawariensis resembles S. aureus in that 
spiral formation occurs with both cultures 
and both form soluble brown pigments in 
organic media. They differ in that S. aureus 
liquefies gelatin to a greater extent. S. ha- 
wariensis 1s also similar to S. bekiniensts in 
some of its cultural properties; both produce 


THE ACTINOMYCETES, Vol. II 


a white aerial mycelium which becomes 
gray-colored; the sporulation of S. hawazien- 
sis takes place in the form of spirals in its 
aerial mycelium, whereas S. bekiniensis is 
completely devoid of spirals and produces an 
alkaline reaction accompanied by hydroly- 
sis in milk. 

Type culture: ATCC 12,236. 

120. Streptomyces hiroshimensis Shinobu, 
1955 (Shinobu, R. Seibutsugakkaishi 6: 43— 
46, 1955). 

Morphology: Sporophores produce verti- 
cils of the Nitella type, both primary and 
secondary. No spirals. Spores elliptical to 
oval,0:8 to 1.24 (Pl. V¥,-Gb). 

Sucrose nitrate agar: Growth poor, re- 
stricted, pink. Aerial mycelium scant, pale 
pink to pinkish-white. 

Calcium malate agar: Growth slow. Aerial 
mycelum pale cinnamon-pink. Soluble pig- 
ment brownish. 

Glucose-asparagine agar: Growth good, 
reddish-pink. Aerial mycelium pink to pur- 
plish-pink. Soluble pigment usually absent, 
sometimes pale brown. 

Nutrient agar: Growth 
Aerial mycelium absent, or scant, pale pink 
to pinkish-white. Soluble pigment brownish- 


reddish-brown. 


orange. 

Starch agar: Growth red to purplish-red. 
Aerial mycelium pink to pale pink. 

Potato plug: Growth deep pinkish-red to 
Aerial mycelium 
pinkish-white. Soluble pigment brownish- 
black. 

Gelatin: Growth pale reddish-brown. Aer- 
pinkish- 


brownish-black. scant, 


ial mycelium absent, or scant, 
white. Soluble pigment pale reddish-brown. 
Rapid liquefaction. 

Milk: Growth deep  pinkish-red. Aerial 
mycelium pinkish-white to pink. Soluble pig- 
ment brown with reddish tinge. No coagu- 
lation; rapid peptonization. 

Nitrate reduction: Strong. 

Starch: Rapid hydrolysis. 

Tyrosinase reaction: Positive. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 229 


Cellulose: Not attacked. 

Carbon utilization: I'ructose and inositol 
well utilized; xylose, rhamnose, sucrose, lac- 
tose, raffinose, and mannitol not utilized; 
galactose and trehalose slightly utilized. 

Antagonistic properties: Inhibits growth 
of gram-positive bacteria and fungi. 

Source: Isolated from soil in Hiroshima, 
Japan. 

temarks: Resembles S. rubrireticuld. 

121. Streptomyces hirsutus Ettlinger et al., 
1958 (Ettlinger, L., Corbaz, R., and Hiitter, 
R. Arch. Mikrobiol. 31: 344, 1958). 

Morphology: Sporophores 
podially branched; short open spirals with 
about three coils are produced. Spores cov- 


long, mono- 


ered with narrow, long spines (PI. II, k). 

Glycerol nitrate agar: Growth colorless. 
Aerial mycelium at first milky white, later 
leek-green. 

Glucose-asparagine agar: Growth color- 
less. No aerial mycelium. 

Glycerol malate agar: Growth at 
milky white; later covered with aerial my- 
celium gradually colored leek-green. 

Starch-KNOs; agar: Growth milky white. 
Aerial mycelium leek-green. Starch hydro- 


first 


lyzed. 

Gelatin: Growth whitish-yellow, covered 
with light green aerial mycelium. Slow lique- 
faction. No soluble pigment. Melanin-nega- 
tive. 

Potato: Growth colorless. Aerial mycelium 
at first white, later leek-green. 

Milk: Pellicle heavy, light yellow. Aerial 
mycelium white-gray. Rapid coagulation; no 
peptonization. 

Antagonistic properties: None. 

Habitat: Soil in Switzerland. 

temarks: Some of the cultures described 
by Gause et al. (1957), such as A. acrimycini 
and A. acrimycint var. globosus, are closely 
related to this organism. 


(Bostroem 
Waksman 


122. Streptomyces hominis 
1890; emend. Waksman, 1919) 


and Henrici, 1948. (Bostroem, E. Beitr. 
pathol. Anat. allgem. Pathol. 9: 1-240, 


1890; Waksman, S. A. Soil Sci. 8: 129-130, 
1919. 

Synonyms: Streptothrix hominis Fouler- 
ton, 1899. Oospora hominis Ridet, 1911. 

Morphology: Sporophores straight. A few 
dextrorse spirals on glycerol synthetic me- 
dia. 

Sucrose nitrate agar: Growth white with 
shade of yellow, turning brownish with age. 
Aerial mycelium white with olive tinge. No 
soluble pigment. 

Glycerol malate agar: Growth yellowish. 
Aerial mycelium with olive-green tinge. 

Nutrient agar: Growth yellowish. Aerial 
mycelium white. No soluble pigment. 

Starch: Hydrolysis good. 

Potato: Growth yellowish to orange, be- 
coming brown. Aerial mycelium white. Color 
of plug unchanged, later becoming brown. 

Gelatin: Growth cream-colored. No aerial 
mycelium. No soluble pigment. 

Milk: Rapid coagulation and peptoniza- 
tion. 

Nitrate reduction: Positive. 

Sucrose: Not inverted. 

Production of HoS: Negative. 

Habitat: Supposed to have been isolated 
from abscess of palm of hand; probably an 
ar contamination. Appears to be related to 
the S. griseus series. 


125. Streptomyces humidus Nakazawa and 
Shibata, 1956 (Belgian Patent 533,386. Ta- 
keda Pharmaceutical Industries Ltd., Ja- 
pan, March 24, 1956; Proc. Japan Acad. 
32: 648-653, 1956). 

Morphology: Sporophores form spirals. 
PANTO Te 
Growth 


Spores oval, 1 to 1.5 by 
nitrate agar: 
Aerial mycelium white. No soluble pigment. 

Nutrient agar: Growth colorless. No aer- 
ial mycelium. No soluble pigment. 


Sucrose colorless. 


Glucose-asparagine agar: Growth color- 
less. Aerial mycelium white to smoke-gray 
or vinaceous-chamois. No soluble pigment. 


230 


Calcium malate agar: Growth colorless, 
becoming yellowish. Aerial mycelium white. 
No soluble pigment. 

Starch agar: Growth colorless to cream- 
colored. Aerial mycelium white to pale 
smoke-gray. No soluble pigment. 

Potato: Growth colorless. Aerial mycelium 
white to smoke-gray; black, moist speckles. 
No soluble pigment. 

Gelatin: Growth colorless. No aerial my- 
celium. No soluble pigment. Moderate lique- 
faction. 

Milk: Growth colorless. Aerial mycelium 
white. No soluble pigment. Slow peptoniza- 
tion. 

Nitrate reduction: Positive. 

Carbon utilization: b-xylose, L-arabinose, 


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Fraure 42. 8S. hygroscopicus (Reproduced from: 
Tresner, H. D. and Backus, EH. J. Appl. Micro- 
biol. 4: 246, 1956). 


THE ACTINOMYCETES, Vol. II 


L-rhamnose, b-fructose, galactose, maltose, 
lactose, D-mannitol, salicin utilized. Sucrose, 
D-raffinose, inulin, p-sorbitol, dulcitol, 2-ino- 
sitol, sodium acetate, sodium citrate not uti- 
lized. 

Antagonistic properties: Produces an anti- 
biotic, acidomycin, said to be dihydrostrep- 
tomycin (see also Imamura et al., 1956). 

Remarks: S. huwmidus is closely related to 
S. hygroscopicus; growth of the latter on 
agar media and on potato is cream-colored 
to yellow to brown. 


124. Streptomyces hygroscopicus (Jensen, 
1951) Waksman and Henrici, 1948 (Jensen, 
H. L. Proc. Linnean Soc. N. 8. Wales 56: 
30/—358, 1931). 

Morphology: Sporophores monopodially 
branched, with narrow compact, sinistrorse 
spirals, situated as dense clusters on the 
main stems of the sporophores (Fig. 42). 
Spores oval, 0.8 to 1.0 by 1:0 to 1:22, 
smooth (PI. II nf, Pl. IV Gb). 
nitrate agar: Growth folded, 
white to cream-colored, later sulfur-yellow 
to yellowish-gray, with golden to light orange 
reverse. Aerial mycelium scant, white to ash- 
gray. Soluble pigment golden to light orange. 

Glucose-asparagine agar: Growth cream- 
colored to straw-yellow, later dull chrome- 
vellow to brownish-orange. Aerial mycelium 


Sucrose 


dusty white to pale yellowish-gray; later 
small, moist, dark violet-gray to brownish 
patches produced, gradually spreading over 
the whole surface. Soluble pigment light 
yellow. 

Nutrient agar: Growth wrinkled, cream- 
colored, later vellowish-gray with yellowish- 
brown reverse. Aerial mycelium scant, white. 

Potato: Growth raised, wrinkled, cream- 
colored, later yellowish-gray to dull brown- 
ish. Aerial mycelium absent or trace of 
white. Melanin-negative. 

Gelatin: Liquefaction slow. No soluble 
pigment. 

Milk: No coagulation; positive peptoniza- 


DESCRIPTION OF SPECIES OF STREPTOMYCES 231 


tion. The reaction becomes faintly acid (pH 
6.0 or less). 

Starch: Hydrolysis. 

Cellulose: Ready decomposition by some 
strains. 

Nitrate reduction: None with sucrose as 
source of energy. 

Sucrose inversion: Positive. 

Production of H.S: Negative. 

Antagonistic properties: Produces hygro- 
mycin, an antibiotic active against mycobac- 
teria and roundworms (see also Pagano ef al., 


1953). 
Habitat: Soil. 
Remarks: Tresner and Backus (1956) 


made a comprehensive study of 18 cultures 
representing S. hygroscopicus and closely re- 
lated forms. They came to the conclusion 
that the following three properties are the 
fundamental the organ- 
ism: (1) sporophores terminate in tight spi- 
rals of a few to many turns, plus a clustering 
of such sporophores along hyphae; (2) 
brownish-gray (mouse-gray to benzo-brown) 


characteristics of 


LD) 


spore color on favorable media; (3) distine- 
tive hygroscopic character on some agar 
media. The characteristic feature, not 
equally distinct in all strains, however, is the 
fact that the aerial mycelium on synthetic 
media becomes moistened and exhibits dark, 
glistening patches; when touched with a 
needle, these patches prove to be moist, 
smeary masses of spores. Shape and size of 
spores, color of substrate growth, formation 
of soluble pigments, growth on potato, 
growth on milk, cellulose decomposition, and 
carbon and nitrogen utilization were con- 
sidered by Tresner and Backus as variable 
properties. They considered S. platensis and 
S. endus as closely related. 

Other related forms, such as A. kurssano- 
vi and A. nigrescens, have been described by 
Gause et al. (1957). Ettlinger et al. (1958) 
also included S. platensis and S. rutgersensis 
var. castelarense in this group. The relation- 
ship of this species to S. violaceoniger has 


been indicated by Nomi (1960b). Vavra et al. 
(1959) described a variety decoyicus that 
differed in certain minor properties. A num- 
ber of other varieties of this organism have 
been described, such as odoratus (Yiintsen 
et al., 1956) and angustmyceticus (Takahashi 
and Amano, 1954). 


125. Streptomyces intermedius (Kriiger- 
emend. Wollenweber, 1922) Waksman (Wol, 
lenweber, H. Ber. deut. botan. Ges. 39: 26, 
1922). 

Morphology: Sporophores straight, wavy, 
frequently arranged in fascicles or clumps 
No spirals. Spores round to oval; 0.9 to 1.0 
by 0.7 uy. 

Glycerol nitrate agar: Substrate growth 
cream-colored to brown; sometimes dark 
green to greenish-brown. Aerial mycelium 
thin, gray to dark gray. 

Glucose-asparagine agar: Growth brown- 
ish with greenish shade. Aerial mycelium 
dark gray. No soluble pigment. 

Nutrient agar: Growth much __ folded, 
cream-colored. Aerial mycelium white. Sol- 
uble pigment faintly golden, occasionally 
green to olive-green; on continued cultiva- 
tion, green color tends to become cream- 
colored to brownish. 

Glucose-peptone Growth good, 
brownish. Aerial mycelium heavy, cream- 
colored to dark gray. No soluble pigment. 

Potato: Growth folded, brown to greenish- 


agar: 


brown. Aerial mycelium dark gray. Soluble 
pigment olive-green. Melanin-negative. 

Gelatin: Growth thin, colorless to faintly 
brown, dropping to bottom. Slow liquefac- 
tion. Greenish-brown pigment. 

Milk: Surface growth heavy, cream-col- 
ored. No aerial mycelium. No coagulation, 
good peptonization. 

Starch: Good hydrolysis. 

Cellulose: Growth good, olive-green. Aer- 
ial mycelium dark gray. 

Sucrose: Inversion slow. 

Nitrate reduction: Limited. 

Habitat: Potato scab. 


~) 
bo 


Remarks: Above description was supple- 
mented by Hoffmann (1958). Krassilnikov 
(1949) considers this organism as a variety of 
A. cretaceus. 

Type culture: IMRU 3329. 


126. Streptomyces tpomoeae (Person and 
Martin, 1940) Waksman and Henrici, 1948 
(Person, L. H. and Martin, W. J. Phyto- 
pathology 30: 913, 1940). 

Morphology: Spores oval to elliptical, 0.9 
to 1.3 by 1.8 to 1.8 yw. 

Sucrose nitrate agar: Growth abundant, 
wrinkled, olive-yellow. No aerial mycelium. 

Nutrient agar: Growth moderate, in form 
of small, shiny colonies, both on the surface 
and imbedded in the medium, silver-colored. 

Starch agar: Growth moderate, smooth, 
ivory-colored. Aerial mycelium white with 
patches of bluish-green. No soluble pigment. 
Complete hydrolysis after 12 days. 

Potato: Growth moderate, shiny, wrin- 
kled, light brown. No aerial mycelium. No 
soluble pigment. 

Gelatin: Growth scanty, after 25 days at 
20°C. No aerial mycelium. No soluble pig- 
ment. Some liquefaction. 

Milk: Ring on surface. No visible coagu- 
lation; positive peptonization. 

Cellulose: No growth. 

Nitrate reduction: Positive. 

Antagonistic properties: Positive. 

Habitat: Lesions caused by sweet-potato 
disease. 


Type culture: IMRU 3476. 


kanamyceticus Okami 
and Umezawa, 1957 (Umezawa, H., Ueda, 
M., Maeda, K., Yagishita, K., Kondo, S., 
Okami, Y., Utahara, R., Osato, Y., Nitta, 
KX., and Takeuchi, T. J. Antibiotics (Japan) 
LOA: 181-188, 1957). 


127. Streptomyces 


Morphology: Sporophores flexible and 
hooked, no true spirals. 
Glycerol nitrate agar: Growth at first 


colorless, later lemon-yellow. Aerial myce- 
lium white to yellow, occasionally with a 


THE ACTINOMYCETES, Vol. II 


greenish or faint pinkish tinge. Soluble pig- 
ment occasionally produced, faint brown. 

Growth color- 
less to yellow with faint pinkish-white, and 
yellow or hay-colored reverse. Aerial myce- 
hum scant; develops from center of colony. 
white to faint pinkish-white to greenish- 


Glucose-asparagine agar: 


yellow or yellow. Soluble pigment occasion- 
ally produced, faint brown. 

Calcium malate agar: Growth yellow. 
Aerial mycelium white-yellow. 

Nutrient agar: Growth cream-colored. 
Aerial mycelium absent or white. No soluble 
pigment. 

Potato: Growth wrinkled, faint yellowish- 
brown to yellow. Aerial mycelium scant, 
white. No soluble pigment. Substrate be- 
neath growth occasionally changes to brown. 

Gelatin: Liquefaction positive. No soluble 
pigment. Melanin-negative. 

Milk: Growth colorless. Aerial mycelium 
absent or white. Coagulation and peptoni- 
zation doubtful. 

Blood agar: Growth wrinkled, grayish- 
reddish-brown. No aerial mycelium. No sol- 
uble pigment. 

Starch: Hydrolyzed. 

Carbon utilization: 
dextrin, fructose, galactose, glycerol, mal- 


Utilizes arabinose, 
tose, mannitol, mannose, raffinose, starch, 
sucrose, and succinate. Does not utilize ino- 
sitol, inulin, lactose, rhamnose, sorbose, xy- 
lose, and acetate. Some strains grow slightly 
on eseulin, salicin, sorbitol, and citrate. 

Antagonistic properties: Produces kana- 
mycin, an antibiotic related to the neomycin 
group. 

temarks: Closely related to S. albido- 


flavus, S. liesket, and S. alboflavus. Okami 


et al. (1959a) made a detailed study of the 
mutants produced by this organism. 


128. Streptomyces kentuckensis Barr and 
Carman, 1956 (Barr, IF. S., and Carman, 
P. I. Antibiotics & Chemotherapy 6: 286— 
289, 1956). 

Morphology: thick- 


Aerial mycelium 


DESCRIPTION OF SPECIES OF STREPTOMYCES 233 


walled, generally not branched; sporophores 
straight; do not form spirals. Spores oblong 
to oval, 0.5 by 0.5 to 1.5 uw. Spores produced 
by fragmentation of the hyphae in substrate 
mycelium are generally smaller than those 
formed from aerial hyphae. 

Nutrient agar: Growth gray to yellow. No 
soluble pigment. Melanin-negative. 

Gelatin: Slow liquefaction. No soluble pig- 
ment. 

Potato: Mycehum gray. Plug darkened. 

Milk: Peptonization positive. 

Nitrate reduction: Positive. 

Production of H.S: Negative. 

Carbon utilization: Readily utilizes vari- 
ous pentoses, hexoses, disaccharides, ace- 
tate and citrate; slight utilization of rham- 
nose, inulin, glycerol, inositol, mannitol; does 
not utilize dextran or salts of oxalic, succinic, 
and salicylic acids. 

Antagonistic properties: Effective against 
gram-positive and some gram-negative bac- 
teria. Produces antibiotic raisnomycin. 

Remarks: Pridham et al. (1958) consider 
this organism as a member of the bzverticilla- 
tus group. 

Type culture: ATCC 12,691. 

129. Streptomyces kimbert (Erikson, 1935) 
Waksman (Erikson, D. Med. Research 
Council (Brit.) Spec. Rept. Ser. No. 203: 
14-15, 1935). 

Morphology: Growth made up of long, 
straight, profusely branching filaments. Ae- 
rial mycelium produces short and straight 
sporophores. Spores small, round. 

Sucrose nitrate agar: Growth 
cream-colored. Aerial mycelium powdery, 
white. 

Glucose-asparagine agar: Growth cream- 
colored. Aerial mycelium white. 

Nutrient agar: Growth moist, cream-col- 
ored. Aerial mycelium powdery, white. 

Gelatin: Colonies smooth, shining, float- 
ing on liquefied medium. Aerial mycelium 
powdery, white. Good liquefaction. No sol- 
uble pigment. 


moist, 


Milk: Coagulation, slow peptonization. 
Surface ring pinkish-brown; medium later 
becomes dark brown. 

Starch: No hydrolysis. 

Source: Blood culture of a woman with 
acholuric jaundice. No record concerning 
actual pathogenicity. 


130. Streptomyces kitasatoensis Hata et al., 
1953 (Hata, T., Koga, F., and Kanamori, 
H. J. Antibiotics (Japan) 6A: 109-112, 
1953). 

Morphology: Sporophores produce pri- 
mary and secondary verticils. A few spirals 
were also found. Spores oval or cylindrical, 
1.9 to 1.3 by 0.9 u. 

Sucrose nitrate agar: Growth yellow to 
light vellowish-brown. Some strains form no 
aerial mycelium even after prolonged culti- 
vation; others form thick grayish-white ae- 
rial mycelium. Soluble pigment light yellow- 
ish. 

Glucose-asparagine agar: Growth brown 
to dark brown, restricted, with raised center. 
Aerial mycelium thin, grayish or mouse- 
grayish. Soluble pigment brown. 

Nutrient agar: Growth brown, restricted, 
with 
Soluble pigment brown. 

Starch Growth 
vellow to yellowish-brown. Aerial mycelium 


raised center. No aerial mycelium. 


agar: colorless; reverse 
yvellowish-white, cottony or flocculent. 

Tyrosine agar: Growth brown to dark 
brown. Aerial mycelium grayish-white, thin, 
later becoming cottony. Tyrosinase reaction 
positive. 

Potato: and 
wrinkled. No aerial mycelium. Color of plug 


Growth  yellowish-brown 
light brown. 

Gelatin: Growth dark brown. Soluble pig- 
ment dark brown. Liquefaction slow at be- 
ginning, but complete liquefaction 4 weeks 
later. 

Milk: No coagulation; slow peptonization. 
No clearing of milk, but heavy brown pre- 
cipitate on bottom; color of liquid in upper 
portion brownish. 


{ THE 


) 


Starch: Hydrolysis positive. 
Cellulose: No decomposition. 
Nitrate reduction: Positive. 
Production of H.S: Positive. 
Carbon utilization: Utilizes 
starch, dextrin, glycerol, galactose, maltose, 


glucose, 


sucrose, trehalose, inositol, sorbitol, sodium 
succinate, sodium citrate, and sodium ace- 
tate. Does not utilize xylose, raffinose, rham- 
nose, lactose, arabinose, mannose, mannitol, 
inulin, dulcitol, fructose, salicin, or esculin. 

Antagonistic properties: Produces an anti- 
biotic, leacomycin. 

temarks: S. kitasatoensis is similar to S. 
reticuli in morphology of the mycelium, cul- 
tural characteristics, and utilization of car- 
bon sources, but different in several other 
respects. 


131. Streptomyces kitasawaensis Harada 
and Tanaka, 1956 (Harada, Y. and Tanaka, 
S. J. Antibiotics (Japan) 9A: 113-117, 1956). 

Morphology: Sporophores straight ; no spi- 
rals. 

Sucrose nitrate agar: Growth cream to 
yellow. Aerial mycelium white with pale 
pinkish tinge. 

Calcium malate agar: Growth cream-col- 
ored. Aerial mycelium white. No soluble 
pigment. 

Glucose-asparagine agar: 
brownish tinge. Aerial mycelium white with 


Growth has 
grayish tinge. Soluble pigment pale yellow- 
ish-brown to pale greenish-yellow. 

Nutrient agar: Growth brownish. Aerial 
mycelium absent or scarce. Soluble pigment 
dark brown. 

Starch agar: Growth pale grayish-brown 
to pale blackish-brown. Aerial mycelium 
white. Soluble pigment pale greenish-yellow 
to pale yellowish-brown. 

Gelatin: Growth white to gray. Aerial my- 
celiuum absent or scarce. Soluble pigment 
dark brown. No or weak liquefaction. 

Potato: Growth brown. Aerial mycelium 
white. Soluble pigment dark brown. 


ACTINOMYCETES, Vol. II 


Milk: Growth in form of dark brownish 
ring. Coagulation and peptonization. 

Carbon utilization: Utilizes p(+)xylose, 
D-mannitol, L-arabinose, salicin. Does not 
utilize L(-++-)rhamnose, b-maltose. 

Antagonistic properties: Produces an anti- 
tumor substance, carzinocidin. 

Habitat: Soil. 


1959 
203, 


132. Streptomyces lanatus Frommer, 
(Frommer, W. Arch. Mikrobiol. 32: 
1959). 

Morphology: Sporophores long, straight 
or wavy, with short side branches; the ends 
of these are more entangled than spiral- 
shaped. 

Glycerol-sucrose agar: Growth abundant, 
cottony, with red-brown reverse. Soluble 
pigment brown to dark red-brown. 

Glucose-asparagine agar: Growth 
brown. Aerial mycelium velvety to cottony, 
rose to gray-green. Soluble pigment brown- 


rose- 


ish. 

Calcium malate agar: Growth colorless to 
yellowish. Aerial mycelium powdery, gray to 
eray-green. No soluble pigment. 

Nutrient agar: Growth yellow-brown. Ae- 
rial mycelium cream-colored or lacking. Sol- 
uble pigment yellow-brown to dark brown. 

Starch: Weak hydrolysis. 

Potato: Growth yellow-brown. Aerial my- 
celium powdery, white. Soluble pigment 
black. 

Gelatin: Growth yellow. Aerial mycelium 
yellow. Soluble pigment brown to red-brown. 
Liquefaction medium. 

Milk: Growth abundant, dark 
Aerial mycelium powdery, cream-colored. 
Coagulation, slow peptonization. 

Cellulose: No growth. 

Antagonistic properties: Produces actino- 


brown. 


mycin. 
femarks: Closely related to S. purpureo- 
chromogenes and S. phaeochromogenes. 


133. Streptomyces lavendulae (Waksman 
and Curtis, 1916) Waksman and Henrici, 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


1948 (Waksman, S. A. and Curtis, R. E. 
Soil Sci. 1: 126, 1916; 8: 130, 1919). 

Morphology: Sporophores long, mono- 
podially branched; short, compact spirals of 
the dextrorse type, 5 to 8 uw in diameter; 
spirals sometimes open. Some strains form 
no spirals, Okami (1956). 
Spores! oval 1:0'sto 2" by “k6to 2:0 
smooth (Pl. V, Ea). 

Sucrose nitrate agar: Growth thin, spread- 
ing, colorless to cream-colored. Aerial my- 


according to 


celium cottony, white, becoming vinaceous- 
lavender. No soluble pigment. 

Glycerol malate agar: Growth cream-col- 
ored. Aerial mycelium lavender. No soluble 
pigment. 

Glucose-asparagine agar: Growth yellow- 
ish. Aerial mycelium white with lavender 
tinge. No soluble pigment. 

Nutrient agar: Growth wrinkled, gray. No 
aerial mycelium. Soluble pigment brown. 

Starch agar: Growth restricted, glistening, 
transparent, rose-colored. Aerial mycelium 
lavender. Good hydrolysis of starch. 

Potato: Growth thin, wrinkled, cream to 
yellowish. No aerial mycelium. Soluble pig- 
ment black. 

Gelatin: Surface growth creamy to brown- 
ish. Aerial mycelium absent or white. Lique- 
faction slow. Soluble pigment brown. 

Milk: Cream-colored ring. No coagulation; 
good peptonization. 

Cellulose: Growth scant. 

Nitrate reduction: Positive. 

Production of H.S: Positive. 

Temperature: Optimum 37°C. 

Antagonistic properties: Various strains 
of this organism produce antibiotics. One 
such antibiotic, streptothricin, is active 
both in vitro and in vivo against various 
gram-positive and gram-negative bacteria, 
fungi, and actinomycetes. Certain other 
strains produce an antiviral agent, ehrlichin. 

Habitat: Soil. 

Remarks: Numerous strains and varieties 
of this organism have been isolated. It is 


235 


sufficient to mention S. lavendulae var. 


japonicus, and several of the cultures listed 


by Gause et al. (1957), notably A. flavotri- 
cint, A. toxytricini, and A. 
linger et al. (1958) considered S. acidomyceti- 


violascens. Ett- 


cus and S. virginiae as members of this 
group. KXrassilnikoy (1949) considered this 
species as a variety of A. chromogenes. 
Okami (1956) and Rangaswami (1958) made 
a detailed study of numerous representatives 
of this species or species-group. 

Morais et al. (1958) described a variety of 
S. lavendulae as brasilicus, the aerial my- 
celium being red-pink or red-brown but not 
lavender and not utilizing salicin. 

Type species: IMRU 3440. 


154. Streptomyces lieskei (Duché, 1934) 
Waksman and Henrici, 1948 (Duché, J. Les 
actinomyces du groupe albus. P. Lechevalier, 
Paris, 1934). 

Morphology: Sporophores form oval 
spores. 

Glucose-asparagine agar: Growth cream- 
colored, later yellowish to green. Aerial my- 
celium white, later yellowish, growing from 
the edge toward the center. Soluble pigment 
dirty yellow to yellow-green. 

Nutrient agar: Growth cream-colored. Ae- 
rial mycelium white. Soluble pigment yellow- 
ish. 

Gelatin: Aerial 
mycelium white. No soluble pigment. Liq- 


Growth cream-colored. 
uefaction rapid. 

Milk: Growth No aerial 
mycelium. Peptonization without coagula- 
tion. After 20 days the whole milk becomes 
a clear yellowish liquid. 

Tyrosine medium: Growth rapid. Aerial 
mycelium whitish-yellow. Soluble pigment 


cream-colored. 


yellowish to orange-yellow. 

Coagulated serum: Growth colorless. Liq- 
uefaction rapid. 

temarks: Related to S. alboflavus and S. 
albidoflavus. 


135. Streptomyces limosus Lindenbein, 


236 THE ACTINOMYCETES, Vol. II 


1952 (Lindenbein, W. Arch. Mikrobiol. 17: 
361-383, 1952). 

Morphology: Substrate mycelium divides 
completely into coccoidal pieces. Some simi- 
larity to Nocardia. Aerial mycelium gray, 
produced in certain media. 

Glycerol nitrate agar: Growth colorless, 
later becoming deep yellow. No aerial my- 
celium. Soluble pigment citron-yellow. 

Glucose-asparagine agar: Growth lemon- 
yellow, later becoming black with yellow 
reverse. Aerial mycelium white, later ash- 
eray. Soluble pigment lemon-yellow. 

Glycerol malate agar: Growth dark yel- 
low. Aerial mycelium white, later ash-gray. 
Soluble pigment golden yellow. 

Nutrient agar: Growth lght brown. No 
aerial Soluble light 
brown. Melanin-positive. 


mycelium. pigment 


Glucose-peptone Growth yellow- 


brown. Aerial mycelium ash-gray. Soluble 


agar: 


pigment yellow-brown. 
Starch-nitrate agar: Growth 
vellow. Aerial mycelium gray-white. Soluble 


brownish- 


pigment light yellow. Hydrolysis strong. 
Potato: Growth brownish-yellow. Aerial 

mycelium Soluble 

lemon-yellow to sulfur-vellow. 
Gelatin: Growth yellow-brown. No aerial 


eray-white. pigment 


mycelium. Soluble pigment dark brown. 
Liquefaction complete. 
Milk: Growth lichenoid, light 


Aerial mycelium gray-white. Soluble pig- 


vellow. 


ment hght brown. Strong peptonization. 
Cellulose: No growth. 
Antagonistic properties: None. 
Source: Isolated from the slime of a river 
bank. 


temarks: Related to S. flavovirens. 


136. Streptomyces ipmani (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 
(Waksman, 8. A. and Curtis, R. E. Soil 
Sci. 1: 123, 1916: 8: 121, 1919). 

Morphology: Sporophores straight, no spi- 
rals. Spores oval, 0.8 to 1.1 by 1.0 to 1.5 wu. 


Sucrose nitrate agar: Growth abundant, 
raised, colorless, becoming light brown and 
wrinkled. Aerial mycelium white, turning 
gray to dark gray. No soluble pigment. 

Glycerol malate agar: Growth colorless, 
dark brown. Aerial 
mouse-gray. No soluble pigment. 


becoming mycelium 

Glucose-asparagine agar: Growth spread- 
ing, light yellow. No aerial mycelium. No 
soluble pigment. 

Nutrient agar: Growth wrinkled, glossy, 
vellow. No aerial mycelium. No soluble pig- 
ment. 

Potato: Growth abundant, wrinkled, 
cream-colored. Aerial mycelium white to 
gray. Soluble pigment purplish. 

Gelatin: Cream-colored, flaky sediment. 
Aerial mycelium white-gray. Liquefaction 
medium to rapid. Melanin-negative. 

Milk: Cream-colored 
and peptonization. 

Starch media: Growth transparent, be- 


ring. Coagulation 


coming dark with age. No aerial mycelium. 
Hydrolysis medium. 

Cellulose: No or very scant growth. 

Invertase: Positive. 

Nitrate reduction: Positive. 

Production of HoS: Negative. 

Temperature: Optimum 25°C. 

Antagonistic properties: Good, though 
some strains show no activity. 

Habitat: Soil. 

femarks: Ettlinger e¢ al. (1958) consider 
this organism as a strain of S. griseus. 
reported that their 
strain produced a grayish-yellow-buff aerial 


Tresner and Danga 
mycelium. Krassilnikov (1949) considered it 
as a variety of A. viridis. 

Type culture: IMRU 3331. 


137. Streptomyces loidensis (Millard and 
Burr, 1926) Waksman (Millard, W. A., and 
Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores. straight and 
spiral-forming. Spores cylindrical to spheri- 
eal, 0.9 to 1.0 by 0.9 to 0.95 wu. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 237 


Sucrose nitrate agar: Growth thin, flat, 
gray to yellowish-olive. Aerial mycelium 
scant, olive-colored. Soluble pigment yellow. 

Nutrient potato agar: Growth good, gray. 
Aerial mycelium olive-buff. Soluble pigment 
golden brown. 

Gelatin: Growth gray. Aerial mycelium 
scant, white. Liquefaction rapid. Soluble 
pigment yellow. 

Milk: Surface growth excellent. Aerial 
mycelium white. Coagulation and rapid pep- 
tonization. 

Starch: Hydrolysis. 

Nitrate reduction: None. 

Temperature: Grows well at 37.5°C. 

Habitat: Potato scab. 


138. Streptomyces longisporoflavus (xras- 
siinikov, 1941) Waksman (Krassilnikov, N. 
A. Actinomycetales. Izvest. Akad. Nauk. 
SSSR, Moskau, p. 30, 1941). 

Morphology: Sporophores produce long 
open spirals. Spores cylindrical or elongated, 
1.0 to 1.5 by 0.7 uw, some rounded at ends 
and swollen in center. 

Agar media: Growth yellow to lemon-yel- 
low or dirty yellow, seldom golden yellow. 
Pigment insoluble. Aerial mycelium well de- 
veloped, velvety, whitish-yellow to brown- 
ish-vellow. 

Gelatin: Liquefaction medium. 

Milk: Coagulation and slow peptonization. 

Starch: Hydrolysis weak. 

Cellulose: No growth. 

Nitrate reduction: Positive. 

Sucrose: No inversion. 

Antagonistic properties: Weakly antago- 
nistic. 

femarks: Some strains, such as S. flavo- 
viridis, sometimes have a greenish or green- 
ish-yellow color instead of a yellow color. 
This organism and related forms belong to 
the same group as S. griseoflavus and S. 
microflavus. The form described by Gause 
ct al. (1957) as A. aurini also belongs to this 
group. 


139. Streptomyces lucensis Arcamone et al., 
1957* (Arcamone, F., Bertazzoli, C., Cane- 
A., Ghione, M., and 
Microbiol. 4: 119-128, 


vazzil, G., DiMarco, 
Grein, A. 
1957). 


Morphology : 


Giorn. 
Aerial hyphae — long, 
branched, and hooked at the tip. Spirals 
produced abundantly. 

Glycerol-glycine agar: Growth  abun- 
dant, lemon-yellow. Aerial mycelium gray- 
brown. Some soluble pigment produced. 

Glucose-asparagine agar: Growth abun- 
dant. Aerial mycelium hazel-brown; scanty 
clusters of white, short, sterile hyphae. No 
soluble pigment. 

Potato-glucose agar: Growth abundant, 
smooth, yellowish. Aerial mycelium abun- 
dant, buff-gray to hazel-brown. Soluble pig- 
ment ash-gray, later turning gray-brown. 

Yeast-glucose agar: Growth dark brown. 
Aerial mycelium whitish. Soluble pigment 
dark brown. 

Starch agar: Growth abundant, colorless 
to yellowish-brown. Aerial mycelium pow- 
dery, buff-gray to light brown. No soluble 
pigment. Moderate starch hydrolysis. 

Oatmeal agar: Growth yellowish and 
smooth. Aerial mycelium hazel-brown. No 
soluble pigment. 

Potato plug: Growth abundant, wrinkled. 
Aerial mycelium light gray to hazel-brown. 
Plug 
culture. 

Gelatin: Growth abundant, brown. Aerial 
turning 


surface turns dark brown around 


mycelium white, erayish-brown. 


Substrate 


) 


is strongly darkened within 3 
days. No liquefaction. 

properties: 
antifungal antibiotic, etruscomycin, of the 


Antagonistic Produces — an 


tetraene type. 

Type culture: IMRU 3783. 

140. Streptomyces luridus  (krassilnikov 
et al., 1957) Waksman (Krassilnikov, N. A., 
Koreniako, A. I., Meksina, M. M., Vale- 


* Supplemented by personal communication. 


238 THE ACTINOMYCETES, Vol. II 


dinskaia, L. K., and Vesselov, N. M. Mikro- 
biologiya 26: 558-564, 1957). 

Morphology: Substrate mycelium mono- 
podially branched, 0.7 to 0.8 uw in diameter. 
Sporophores produce spirals with 1 to 3 
turns. Spores spherical, oval, seldom elon- 
gated. Sporulation is generally weak, oc- 
curring only on certain media; spiral forma- 
tion occurs seldom, largely on synthetic 
media with a limited amount of sucrose, and 
on starch media. 
nitrate agar: 
orange. Aerial mycelium white with rose 


Sucrose Growth  yellow- 
tinge. 

Nutrient agar: Substrate growth colorless, 
free of aerial mycelium. No soluble pigment. 
In old cultures, clumps of aerial hyphae may 
be formed. 

Potato agar: Growth yellow-orange. Aerial 
mycelium white with rose tinge. Crystals of 
salts deposited along the mycelium in the 
substrate. 

Milk: Coagulation weak; rapid peptoniza- 
tion. 

Gelatin: Not liquefied in 10 days. 

Starch: Moderate hydrolysis. 

Nitrate: Reduced. 

Sucrose: Not inverted. 

Cellulose: No growth. 

Carbon utilization with acid formation: 
arabinose, inositol, sorbitol; no acid from 
elucose, lactose, rhamnose, xylose, inulin, 
inositol, mannitol, or dulcitol. 

Antagonistic properties: Produces anti- 
bacterial and antiviral (uridin) substances. 

Yemarks: Cannot be distinguished from 
S. fradiae in its morphological and cultural 
properties, but is different in its biochemical 
and its antibiotic activities. 


141. Streptomyces luteoverticillatus Shin- 
obu, 1956 (Shinobu, R. Mem. Osaka Univ. 
B (N.S.) 5: 84-93, 1956). 
Primary secondary 


Morphology: and 


verticils produced on various synthetic 


media. Spores coccoid to elliptical, about 0.8 


u long. 


Sucrose nitrate agar: Growth pale brown. 
Aerial mycelium cottony, brownish-white 
to brown. 

Glucose-asparagine agar: Growth thin, 
brown. Aerial mycelium cottony, yellow- 
white. Soluble pigment pale brown. 

Nutrient agar: Growth excellent, deep 
brown. Aerial mycelium white to yellow to 
green. Soluble pigment deep brown. 

Potato plug: Growth heavy, brown. Aerial 
mycelium yellow to greenish. Soluble pig- 
ment brown. 

Milk: Aerial mycelium heavy, brown. 
Coagulation uncertain; peptonization strong. 
Soluble pigment brown. 

Gelatin: Strong liquefaction. 

Diastase reaction: Strong. 

Tyrosinase reaction: Strong. 


Carbon utilization: Fructose, mannitol, 
and inositol utilized. Xylose, rhamnose, 


sucrose, lactose, and raffinose not utilized. 

Habitat: Soil. 

Remarks: Resembles S. verticillatus. 

142. Streptomyces lydicus DeBoer et al., 
1955 (DeBoer, C., Dietz, A., Silver, W. S., 
and Savage, G. M. Antibiotics Ann. 1955- 
1956, p. 886-892). 

Morphology: Sporophores long, slightly 
coiled at tip. Spores spherical to oval. 

Sucrose nitrate agar: Substrate growth 
buff-colored. Aerial mycelium white. 

Nutrient agar: Some substrate growth. 
No aerial mycelium. Soluble pigment yellow- 
ish. 

Casein-yeast extract-beef agar: Growth 
olive-tan. Aerial mycelium gray-white with 
flecks of black. Soluble pigment olive-tan. 

Glucose-peptone agar: Aerial mycelium 
eray-white. Soluble pigment vellow. 

Starch agar: Growth good. Aerial myce- 
lium pink-gray-white. Hydrolysis good to 
excellent. 

Gelatin: Some growth. No aerial myce- 
lium. Liquefaction positive. Soluble pigment 


olive-colored. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 239 


Milk: Ring around surface. Peptonization 
positive. 

Nitrate reduction: Positive. 

Carbon utilization: Most and 
organic acids utilized, but not rhamnose, 
inulin, dulecitol, mositol, or the sodium salts 
of formic, oxalic, tartaric, and salicylic acids. 

Production of H.S: Negative. 

Antagonistic properties: Produces an anti- 


sugars 


biotic, streptolydigin, active against various 
bacteria. 
Remarks: Related to S. diastaticus. 


143. Streptomyces macrosporeus KEttlinger 
et al., 1958 (Ettlinger, L., Corbaz, R., and 
Hiitter, R. Arch. Mikrobiol. 31: 346, 1958). 

Morphology: Sporophores monopodially 
branched along the whole axis with open, 
irregular spirals. Spores large, 1.7 to 2 by 
1.5 to 2 uw; short spines (Plate II m). 

Glycerol nitrate agar: Substrate growth 
yellow. Aerial mycelium white-yellow to ash- 
gray. Soluble pigment golden yellow. 

Glucose-asparagine agar: Growth milk- 
white. No aerial mycelium. Soluble pigment 
whitish-yellow. 

Calcium malate agar: Growth yellow. 
Aerial mycelium white-yellow to ash-gray. 
Soluble pigment white-yellow. 

Starch agar: Growth light yellow. Aerial 
mycelium white-gray. Good hydrolysis of 
starch. 

Potato: Growth abundant, light yellow to 
golden yellow. Aerial mycelium ash-gray. 

Gelatin: Growth sparse. Liquefaction 
slow. No soluble pigment. 

Milk: Pellicle light yellow 
brown. Aerial mycelium white to white-gray. 
Coagulation strong; no peptonization. 


to yellow- 


Antagonistic properties: Produces an 
antibiotic, carbomycin. 


Habitat: Soil in Madras, India. 


144. Streptomyces maculatus (Millard and 
Burr, 1926) Waksman and Henrici, 1948 
(Millard, W. A. and Burr, 8. Ann. Appl. Biol. 
13: 580, 1926). 


Morphology: Growth tough, shiny, carti- 
laginous. Aerial mycelium rarely produced, 
though in certain strains it may frequently 
occur, especially when grown on organic 
media. Sporophores straight, short. Spores 
spherical, 0.5 to 0.6 wu. 

Sucrose nitrate agar: 
yellow to orange-red; as the culture ages it 


Growth orange- 
may change to dark green or black. No 
aerial mycelium. 

Nutrient potato agar: Growth vinaceous- 
tawny. Soluble pigment vinaceous-tawny. 

Potato: Growth restricted, raised, pinkish. 
Aerial mycelium scant, white. Soluble pig- 
ment gray to brown. 

Gelatin: Growth 
slow. 

Milk: Growth shght. No coagulation; no 
peptonization. 

Starch: Hydrolyzed. 

Nitrate reduction: Negative. 


limited. Liquefaction 


Tyrosinase reaction: Negative. 

Oxygen requirement: Said to grow well 
under anaerobic conditions. 

Paraffin: Not utilized. 

Temperature: Grows well at 37.5°C. 

Habitat: Potato scab and soil. 

Type culture: IMRU 3376. 


145. Streptomyces madurae — (Vincent, 
1894) nov. comb. (Vincent, H. Ann. inst. 
Pasteur 8: 129, 1894). 

Synonyms: N. babiensis, N. brumpti, N. 
madurae, and N. salmonicolor (A. salmoni- 
color Millard and Burr, 1926). Baldacci (1944) 
listed 17 synonyms. 

Strains of this organism were reported by 
recently by 
Gonzalez Ochoa and Sandoval (1951), to 


various investigators, most 
form, under certain conditions of culture 
and on certain media, such as grain, an 
aerial mycelium, with straight or spiral- 
shaped sporophores; the spores were cylindri- 
cal or oval. This led them to consider this 
organism as a Streptomyces. Mariat (1957) 
was also of the same opinion. Mackinnon 
and Artagaveytia-Allende (1956) consider 


240 


the generic position of this species as far 
from settled. 

Morphology: Growth red to red-brown or 
pink. In tissues it forms granules consisting 
of radiating actinomycosis. Initial branched 
mycelium is said to be nonsegmented. Not 
acid-fast. Aerial mycelium white and pink 
in color. 

Glucose-asparagine agar: Growth cream- 
colored. Some cultures give reddish pig- 
mentation. 

Protein media: Growth good, pinkish. 
Soluble pigment brown. 

Gelatin: Growth glistening, at first white, 
then buff to rose or crimson. Soluble pigment 
irregular and unpredictable, occasionally red. 
Gelatin slowly liquefied. 

Milk: No change, or shg¢ht; coagulation 
slow, if any; peptonization slow. 

Carbon utilization: Utilizes starch, glu- 
cose, mannitol, and xylose, but not lactose 
or paraffin. 

Nitrate reduction: Positive. 

Pathogenicity: This property was vari- 
ously reported. Topley and Wilson (1946) 
stated that this organism causes a local 
tissue disease when inoculated under the skin 
in guinea pigs. Often reported as not patho- 
genic for the usual laboratory animals; 
pathogenic for monkeys. 

Source: Wide geographical distribution. 
Madura foot and other substrates. 


146. Streptomyces marginatus (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, 8S. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight. Spores 
oval to spherical, 0.9 by 0.8 uw. 

Sucrose nitrate agar: Growth thin, echi- 
nate. Aerial mycelium. olive-buff. Soluble 
pigment cream-colored. 

Nutrient 
gray. Aerial mycelium white to whitish- 


potato Growth heavy, 


agar: 
yellow. Soluble pigment light golden brown 
to deep golden brown. 

Potato: Growth good, raised. Aerial myce- 


THE ACTINOMYCETES, Vol. II 


lium abundant, buff to olive-buff. Plug at 
first gray, later becoming black. 

Gelatin: Growth thin, pale olive-gray. 
Aerial mycelium abundant, pale gray to 
olive-buff. Soluble pigment buff. Liquefac- 
tion rapid. 

Milk: Growth Aerial mycelium 
white. Coagulation and peptonization. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Temperature: Grows well at 387.5°C 

Habitat: Potato scab. 


good. 


147. Streptomyces marinolimosus (Zobell 
and Upham, 1944) Waksman (ZoBell, C. E. 
and Upham, H. C. Bull. Scripps Inst. 
Oceanogr. Univ. Calif. 5: 239-292, 1944). 

Morphology: Aerial mycelium consists of 
branching filaments with chains of spores. 
Spores 0.9 uw in diameter. 

Agar media: Growth dull. Aerial myce- 
lium white to pinkish, powdery, rough, in 
concentric circles. Odor of freshly turned 
soil. 

Potato: Growth yellow, becoming white, 
powdery, raised, rugose. Potato darkened. 

Gelatin: Growth in form of flat, yellowish, 
circular colonies, with tendency to grow 
together. Liquefaction crateriform. Melanin- 
negative. 

Sea water broth: Growth in form of light 
yellow clumps; pellicle produced on surface. 
Earthy odor. 

Milk: Growth in form of pellicle. Com- 
plete peptonization in 20 days. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Production of H.S: Positive. 

Source: Marine mud. 

Remarks: All differential media were pre- 
pared with sea water. 


148. Streptomyces marinus (Humm_ and 
Shepard, 1946) nov. comb. (Humm, H. J. 
and Shepard, IK. S$. Duke Univ. Marine Sta. 
Bull. 3: 77, 1946). 

Morphology: Sporophores sometimes form 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


loose spirals. Spores spherical to oval, 0.8 
to 1.2 u, produced as a dark gray area in 
center of colonies. 

Agar media: Growth sparingly branched, 
dense, entangled, frequently forming con- 
centric rings In response to alternate periods 
of light and Aerial mycelium 
white. Spores gray to dark gray. No soluble 
pigment. 

Gelatin: Growth arborescent. Liquefaction 
stratiform, slow. Melanin-negative. 

Milk: Peptonization 
within 1 month. 

Starch: Vigorous hydrolysis. 

Cellulose: Not attacked. 

Chitin and alginic acid: Attacked. 

Agar: Slowly digested, softened, not lique- 
fied. Growth on agar in culture dish sur- 


darkness. 


complete, usually 


rounded by rather wide, gently sloping 
depression. Gelase field relatively wide, with 
distinct margin. Irish moss and Hypnea gels 
also slowly digested. 

Nitrate reduction: Usually negative. In 
some media, slight nitrite is produced after 
10 days’ incubation, especially if glucose is 
present. 

Production of H.S: Positive. 

Indole: Not formed. 

Carbon utilization: Acid produced from 


galactose, glucose, fructose, mannose, cel- 
lobiose, lactose, maltose, sucrose, and 
glycerol. Arabinose, xylose, rhamnose, and 


sorbitol utilized without acid production. 
No growth with raffinose, salicin, inulin, 
dulcitol, inositol, ethyl alcohol, or ethylene 
glycol. Utilizes acetic, citric, lactic, propi- 
onic, succinic, and 7so-valeric acids. Does not 
utilize butyric, gluconic, maleic, malonic, 
and oxalic acids. 
Habitat: Marine sediments. 


149. Streptomyces mashuensis Sawazaki 
et al., 1955 (Sawazaki, T., Susuki, 8., Naka- 
mura, G., Kawasaki, M., Yamashita, S., 
Isono, K., Anzai, K., Serizawa, Y., and 
Sekiyama, Y. J. Antibiotics (Japan) 8A: 
44-47, 1955). 


241 


Morphology: Sporophores straight, no 


spirals; numerous primary and secondary 
verticils. 
Sucrose Growth 


nitrate yellow; 


reverse yellow-green. Aerial mycelium abun- 


agar: 


dant, powdery. 
Glucose-asparagine agar: Growth pow- 
dery, grayish-white, reverse yellow-brown. 
No aerial mycelium. 
Nutrient 
brown. 


agar: Growth cream-colored; 


reverse No. aerial 


soluble pigment. 


mycelium. No 


Starch agar: Growth marguerite-colored ; 
margin cottony, primrose-pink; reverse yel- 
low-brown, margin white. Aerial mycelium 
white. Strongly diastatic. 

Potato: Growth spreading, dark cream- 
colored. Aerial mycelium limited. Limited 
discoloration of plug. 

Gelatin: Growth white. No aerial myce- 
lum. Soluble pigment pinkish. Liquefaction 
medium. 

Nitrate reduction: Negative. 

Carbon utilization: Nylose, glucose, su- 
crose, trehalose utilized. Rhamnose, raffi- 
nose, salicin, mannitol, lactose, arabinose 
not utilized. 

Antagonistic properties: Produces two 
antibiotics, streptomycin and a labile sub- 
stance active against mycobacteria, fungi, 
and B. subtilis. 

Remarks: Okami et al. (1959b) made a 
detailed study of this organism. They re- 
ported, imstead of the yellow growth on 
synthetic media, poor colorless growth. 


150. Streptomyces matensis Margalith et 
al., 1959 (Margalith, P., Beretta, G., and 
Timbal, M. T. Antibiotics & Chemotherapy 
9: 71-75, 1959). 

Morphology: Sporophores produce verti- 
cils, the branches forming spirals. Spores 
spherical. 

Sucrose nitrate agar: Growth colorless, the 
reverse being hyaline to light violet-gray. 
Aerial mycelium powdery, gray. Faint 
bluish pigment. 


242 


Glucose-asparagine agar: Growth hyaline 
with pinkish reverse. Aerial mycelium pres- 
ent. No soluble pigment. 

Calcium malate agar: 
aerial mycelium. 

Nutrient agar: Growth abundant, color- 
less. Aerial mycelium whitish. Soluble pig- 


Growth poor. No 


ment amber. 

Starch: Strong hydrolysis. 

Potato: Growth abundant. Aerial myce- 
lium light gray. No soluble pigment. 

Gelatin: Partial liquefaction. No soluble 
pigment. 

Milk: No coagulation; some peptoniza- 
tion. 

Nitrate reduction: Negative. 

Cellulose: Good growth. 

Carbon utilization: Utilizes glucose, su- 
crose, lactose, galactose, rhamnose, xylose, 
inositol, sodium succinate, and others. Does 
not utilize sucrose, raffinose, glycine, or 
sodium citrate. 

Antagonistic properties: Produces an anti- 
bacterial agent, matamycin. 

Habitat: Soil. 

Remarks: Related to S. noboritoensis and 
S. spiralis. 

Characteristic properties: Culture said to 
be melanin-positive; it produces gray to 
violet spore masses. 


151. Streptomyces mediocidicus Okami et 
al., 1954 (Okami, Y., Utahara, R., Naka- 
mura, 8., and Umezawa, H. J. Antibiotics 
(Japan) 7A: 98-1038, 1954). 

Morphology: Aerial mycelium sometimes 
produces verticils, depending on composition 
of medium; no spirals. 

Glycerol nitrate agar: Growth colorless to 
yellowish. Aerial mycelium absent, or white 
patches. Soluble pigment absent, or slightly 
yellowish brown. 

Glucose-asparagine agar: Same as above. 

Starch agar: Same as above. Diastatic 
action weak or medium. 

Nutrient agar: Growth colorless or shghtly 


THE ACTINOMYCETES, Vol. II 


yellowish. No aerial mycelium. Soluble pig- 
ment brownish. Melanin-positive. 

Potato: Growth yellowish or light yellow- 
ish-brown. Aerial mycelium absent or white. 
No soluble pigment. 

Gelatin: Growth yellowish-brown. No 
aerial mycelium. Soluble pigment brown. 
Strong liquefaction. 

Blood agar: Growth yellowish-brown to 
reddish-brown. Aerial mycelium absent or 
white. Hemolytic action strong. 

Milk: Surface ring colorless to yellowish. 
No aerial mycelium. Soluble pigment 
shghtly brownish. Slow coagulation and 
peptonization. 

Nitrate: No reduction. 

Antagonistic properties: Produces an anti- 
fungal substance, mediocidin, a polyene of 
the hexaene type. 

Type culture: IMRU 3777. 


152. Streptomyces melanocyclus (Merker, 
1911, emend. IWrainsky) Waksman and 
Henrici, 1948 (Merker, E. Centr. Bakteriol. 
Parasitenk. Abt. II, 31: 589, 1912; Krainsky, 
A. ibid. 41: 649-688, 1914). 

Morphology: Spores spherical, 0.9 uw. 

Agar media: Growth much folded, red. 
Aerial mycelium dark brown. Soluble pig- 
ment dark brown, turns whole culture red- 
brown to almost black with a shade of red. 

Calcium malate agar: Colonies small, flat, 
orange-red. Aerial mycelium black, occur- 
ring along the edges. 

Gelatin: Growth poor. Liquefaction rapid. 

Milk: Coagulation and rapid peptoniza- 
tion. 

Starch: Hydrolysis. 

Cellulose: Good decomposition; black cir- 
cles produced on paper. 

Nitrate reduction: Positive. 

Sucrose: Inverted. 

Pigment: Insoluble in water and in or- 
ganic solvents. Considered by Kriss to be 
related to the melanins. 

Strong effect 


Antagonistic properties: 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


upon various bacteria; some strains show 
no activity. 
Habitat: Soil. 
Remarks: A. 
1914) and A. 
1928) are related to above species. 


melanosporeus (IXrainsky, 


melanogenes (Rubentschik 


1538. Streptomyces melanogenes Sugawara 
and Onuma, 1957 (Sugawara, R. and 
Onuma, M. J. Antibiotics (Japan) LOA: 138— 
Hee 1957) 

Morphology: Sporophores monopodially 
branched; no spirals, sometimes slight cur- 
vature. Spores cylindrical, 1.7 to 0.8 by 
0.9 to 0.5 up. 

Sucrose nitrate agar: Growth moist, fold- 
ing, colorless to grayish-red-brown; reverse 
yellow-orange. Aerial mycelium thin, brown- 
ish-white. Soluble pigment brownish-yellow. 

Glucose-asparagine agar: Growth colorless 
to cream-colored with dark reddish center; 
reverse dark yellow-orange. Aerial my- 
celium pale grayish-white. Soluble pigment 
yellowish-brown. 

Calcium malate agar: Growth colorless 
to brownish-yellow to grayish-blue-black. 
Aerial mycelium yellow-white. Soluble pig- 
ment greenish-yellow to brown. 

Nutrient agar: Growth cream-colored to 
brown. No aerial mycelium. Soluble pigment 
reddish-brown. 

Potato: Growth folded, colorless to yel- 
Aerial mycelium 
Soluble pigment 


brownish 


dark 


lowish-brown. 
or grayish-white. 
yellowish-brown. 

Gelatin: Growth colorless to dark brown. 
Aerial mycelium white to gray. Soluble pig- 
ment pale yellowish-brown. Liquefaction 
weak. 

Milk: Cream-colored to dark brown ring. 
Soluble pigment pinkish-brown. 

Blood agar: Growth glistening, vellowish- 
gray to dark olive-gray. No aerial mycelium. 
Soluble pigment dark brown. Hemolysis 
positive. 

Antagonistic properties: Produces a mela- 
nin-like tumor-inhibiting substance. 


243 


Remarks: Resembles S.  phaeochromo- 
genes, S. griseocarneus, and S. cinnamonen- 


SUS. 


154. Streptomyces michiganensis Corbaz 
et al., 1957 (Corbaz, R., Ettlinger, L., 
Keller-Schierlein, W., and Zahner, H. Arch. 
Mikrobiol. 26: 192-208, 1957). 

Morphology: Sporophores straight, ar- 
ranged in sympodially branched clusters; 
no spirals. Spores smooth (Pl. II i). 

Glycerol nitrate agar: Growth whitish- 
yellow. Aerial mycelium velvety, white to 
yellowish to greenish-gray. 

Calcium malate agar: Growth thin, golden 
vellow. Aerial mycelium chalk-white, be- 
coming light yellow. 

Glucose-asparagine agar: Growth thin, 
white to yellow, changing to light yellow- 
red. Aerial mycelium velvety, white-yellow. 

Glucose-peptone agar: Growth wrinkled, 
at first hght brown, then copper-red, finally 


reddish-brown. Aerial mycelium velvety, 
ereenish-gray. Soluble pigment  reddish- 


brown. 

Gelatin: Pellicle ight brown. Aerial my- 
celium powdery, chalk-white. Liquefaction 
slow. Soluble pigment brown. 

Starch: No hydrolysis. 

Potato: Growth light yellow. Aerial my- 
celium velvety, white-gray to white-yellow. 
Soluble pigment gray-black. 

Milk: Pellicle light brown. Aerial myce- 
lium sparse. Coagulation and peptonization. 

Tyrosinase reaction: Positive. 

Antagonistic properties: Produces actino- 
mycin X. 

Carbon utilization: Xylose, 
fructose, galactose, maltose, mannitol, salicin 


arabinose, 


utilized. Rhamnose, sucrose, lactose, raffi- 
nose, inulin not utilized. 
Habitat: Soil. 


155. Streptomyces microflavus (KXrainsky, 
1914) Waksman and Henrici, 1948 (Krain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
II., 41: 686, 1914). 


244 THE ACTINOMYCETES, Vol. II 


Morphology: Spores spherical to  rod- 
shaped, often produced in pairs or in chains, 
2-Ov byt Zo aa: 
Calcium malate agar: Colonies minute, 
yellow. No aerial mycelium. 
Glucose-asparagine Aerial 
lium produced late (12 days), rose-yellow. 
Nutrient agar: Colonies yellow. Aerial 
mycelium produced late, yellowish-rose. 
Potato: Growth yellowish, slimy mass. 
No aerial mycelium. Melanin-negative. 
Gelatin: Colonies small, yellowish. Lique- 


agar: myce- 


faction rapid. 

Milk: Rapid coagulation and peptoniza- 
tion. 

Invertase: Negative. 

Starch: Diastatic action strong. 

Cellulose: Growth scant, white. 

Nitrate reduction: Positive. 

Production of HS: Negative. 

Antagonistic properties: Said to produce 
a form of streptothricin. 

Habitat: Soil. 

Remarks: According to Ettlinger et al. 
(1958), this organism belongs to the S. 
griseus series. 

Type culture: IMRU 3332; ATCC 13,281. 


156. Streptomyces mirabilis Ruschmann, 
1952 (Ruschmann, G. Pharmazie 7: 542- 
550, 639-648, 823-831, 1952). 

Morphology: Sporophores straight, with- 
out spirals or curvature. 

Agar media: Aerial mycelium white, 
cottony. 

Nutrient agar: Growth 
slimy surface. No aerial mycelium. 


Glucose agar: Growth grayish-brown. No 


poor, forming 


aerial mycelium. Soluble pigment brown. 

Potato: Growth good, lichenoid. Soluble 
pigment dark brown to black. 

Gelatin: Good flaky growth. Rapid lique- 
faction. Soluble pigment dark brown to 
black. 

Milk: Surface growth covered with white, 
fluffy Coagulation and 


aerial mycelium. 


peptonization positive. Liquefied 
colored black. 
Fats: Ready utilization. 


Temperature: Optimum 29°C. No growth 


portion 


(> ay AO} 


at o¢ 

Antagonistic properties: Antagonistic ef- 
fect strongest in freshly isolated cultures. 
Property lost on cultivation; activity lost 
first against gram-negative, rod-shaped bac- 
teria, coccl remaining most sensitive. Pro- 
duces antibiotic miramycin. 

temarks: Highly proteolytic and lipoly- 
tic. Grows best on complex organic media, 
at slightly acid reaction, pH 6.0 to 6.6. 


157. Streptomyces mitakaensis Arai et al., 
1958 (Arai, M., Karasawa, K., Nakamura, 
S., Yonehara, H., and Umezawa, H. J. 
Antibiotics (Japan) 11A: 14-20, 1958). 

Morphology : Sporophores short, 
branched; spirals produced. Spores spheri- 
eal, 1.2: to 1.5: a: 

Sucrose nitrate agar: Growth good, color- 
less or white to dark yellowish-brown. Aerial 
mycelium powdery, abundant, light gull- 
eray. No soluble pigment. 

Glucose-asparagine agar: Growth good, 
white to brown; becomes dark 
brown. Aerial mycelium powdery, abundant, 
gray. No soluble pigment. 

Glycerol citrate Growth 
colorless or white to brownish-white; later 
brownish-yellow. Aerial mycelium powdery, 
abundant, whitish-gray in center, gray in 
the edges. No soluble pigment. 

Nutrient agar: Growth good, colorless to 


reverse 


agar: good, 


pale yellowish-brown. Aerial mycelium pow- 
dery, abundant, white, with or without 
eray parts. No soluble pigment. 

Starch agar: Growth good, colorless to 
pale yellowish-brown. Aerial mycelium pow- 
dery, abundant, light gray in center, gray 
at the edges. No soluble pigment. Hydroly- 
sis strong. 

Potato plug: Growth 
eream-buff. Aerial mycelium poorly devel- 


good, wrinkled, 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


oped, white or grayish-white. No soluble 
pigment. 

Gelatin: Growth colorless. Aerial myce- 
lium pale gull-gray. Soluble pigment absent, 
yellow. Liquefaction moderate. 

Milk: Growth good, colorless to white. 
Peptonization and coagulation rapid. 

Nitrate reduction: Negative. 

Antagonistic properties: Produces — an 
antibiotic, mikamycin, active against gram- 
positive and acid-fast bacteria. 


158. Streptomyces = murinus Frommer 
(Frommer, W. Arch. Mikrobiol. 32: 198, 
1959). 

Morphology: Sporophores small, mono- 
podially branched, tree-like; spirals com- 
pact, with 1 to 3 turns. 

Glycerol nitrate agar: Growth greenish- 
yellow. Aerial mycelium thin, white. Soluble 
pigment greenish-yellow. 

Glycerol-glycine yellow 
to brown-yellow. Aerial mycelium white- 
gray to gray-brown. Soluble pigment golden 


agar: Growth 


yellow. 

0 Growth yellow, 
occasionally brown-violet. Aerial mycelium 
powdery white to gray-white. Soluble pig- 


Glucose-asparagine agar: 


ment yellow to greenish-yellow. 

Calcium malate agar: Growth colorless. 
Aerial mycelium white. No soluble pigment. 

Nutrient agar: Growth yellow to green- 
ish-yellow. Aerial mycelium lacking or 
white. Soluble pigment lacking or golden 
yellow. 

Starch media: Growth colorless to yellow- 
ish. Aerial mycelium gray-brown. No hy- 
drolysis after 10 days. 

Potato: Growth abundant, golden brown. 
Aerial mycelium cream-colored to yellow. 
Soluble pigment questionable. 

Gelatin: Growth abundant, golden yellow. 
Aerial mycelium eray. 
Soluble pigment yellow to golden yellow. 
Liquefaction limited. Melanin-negative. 

Milk: Growth abundant, golden yellow to 


cream-colored to 


245 


Aerial mycelium powdery, 


cream-colored. Questionable coagulation and 


vellow-brown. 


liquefaction. 
Cellulose: Growth weak. Aerial mycelium 
gray-brown. Soluble pigment yellowish. 
Antagonistic properties: Produces actino- 
mycin. 


159. Streptomyces naganisht Yamaguchi 
and Saburi, 1955 (Yamaguchi, T. and 
Saburi, Y. J. Gen. Appl. Microbiol. 1: 201- 
230 L955): 

Morphology: Sporophores straight with 
many compact spirals and a few open 
spirals; spores oval to short rods, 0.8 to 1.4 
by 0.5 to 0.7 up. 

Sucrose nitrate agar: Growth colorless, 
thin. Aerial mycelium powdery, at first 
white, later colored buff. No soluble pig- 
ment. 

Calcium malate agar: Growth is at first 
pinkish-white to pinkish-gray, later becom- 
ing whitish-brown. Aerial mycelium whitish. 
Soluble pigment light pink, but soon disap- 
pears. 

Nutrient agar: Growth at first colorless 
to dark cream, later becoming yellowish- 
brown to brown. No aerial mycelium. Solu- 
ble pigment light brown. Melanin-negative. 

Starch agar: Growth colorless to creamy 
with reddish-purple portion. Aerial myce- 
lium abundant, white or smoke-gray to light 
drab. Soluble pigment absent or faint pink. 
Good hydrolysis. 

Potato: Growth vigorous, at first yellow- 
ish-gray. Aerial mycelium white to grayish- 
white. Soluble pigment deep purple to black. 

Gelatin: Growth dark brown with some 
tint of olive. Soluble pigment deep brown 
and a more diffusible yellowish-green. Lique- 
faction moderate. 

Milk: Growth vigorous, yellowish-brown, 
with white aerial mycelium along the glass. 
Soluble pigment hght 
reddish-brown. Coagulation and peptoniza- 


brown, sometimes 


tion. 


246 


Carbon utilization: Utilizes p-xylose, 
L-arabinose, L-rhamnose, D-galactose, lactose, 
raffinose, mannitol, inositol, salicin, acetate, 
citrate, and succinate; does not utilize su- 
crose, inulin, sorbitol, or cellulose. 

Antagonistic properties: Active against 
gram-positive and acid-fast bacteria, fung), 
and trichomonads. 

temarks: Related to S. antimycoticus. 


160. Streptomyces narbonensis Corbaz_ et 
al., 1955 (Corbaz, R., Ettlinger, L., Gau- 
mann, E., Keller, W., Kradolfer, F., Ky- 
burz, E., Neipp, L., Prelog, V., Reusser, R., 
and Zahner, H. Helv. Chim. Acta 38: 935- 
942, 1955). 

Morphology: Sporophores 
spirals. Spores smooth, cylindrical, 0.8 to 
Lil byaO2760.0.9 ju: 

Glycerol nitrate agar: Growth thin, color- 
less to yellowish-brown. Aerial mycelium 
velvety, whitish-gray. No soluble pigment. 

Glucose-asparagine agar: Growth thin, at 
first colorless, then yellowish-brown. Aerial 
mycelium sparse, chalk-white. No soluble 


straight; no 


pigment. 


Calcium malate agar: Growth colorless. 


as 


FiGguRE 43. isolate AA 877, re- 
sembling S.nelropsis, showing character of verticils 


Hyphae of 


of sporogenous branches (Reproduced from: Dug- 
gar, B. M. etal. Ann. N. Y. Acad. Sei. 60: 85, 1954). 


THE ACTINOMYCETES, Vol. II 


Aerial mycelium white-gray. No soluble 
pigment. 


Glucose-peptone agar: Growth light yel- 
lowish, punctiform. Aerial mycelium pro- 
duced late, powdery, gray-white. No soluble 
pigment. 

Nutrient agar: Growth punctiform, yel- 
lowish. No aerial mycelium. No soluble 
pigment. Melanin-negative. 

Starch agar: Growth thin, colorless to 
vellowish. Aerial mycelium powdery, white. 
No soluble pigment. Hydrolysis good. 

Gelatin: Growth yellowish-white. Aerial 
mycelium snow-white. Soluble pigment light 
reddish-brown. Liquefaction slow. 

Potato: Growth lichenoid, bluish-gray to 
reddish-gray. No aerial mycelium. Soluble 
pigment dark brown. 

Milk: Surface ring whitish-yellow. Pep- 
tonization without coagulation. 

Production of H.S: Positive. 

Antagonistic properties: Produces basic 
antibiotic, narbomycin, related to picromy- 
cin. 

Carbon utilization: Utilizes xylose, arabi- 
nose, rhamnose, fructose, galactose, sac- 
charose, maltose, raffinose, inulin, salicin, 
sodium acetate. Does not utilize mannitol, 
sorbitol, dulcitol, mesoinositol. 

Habitat: Soil. 

femarks: Ettlinger et al. (1958) consider 
this organism as belonging to S. olzvaceus. 


161. Streptomyces netropsis Finlay and 
Sobin, 1952 (Finlay, A. C. and Sobin, B. A. 
U.S. 2.586.762. 1952). 

Morphology: Sporophores in form of ver- 
ticils or terminal clusters on tips of short 
hyphae (Fig. 43). Spores short, cylindrical, 
0.7 by 1.8 uw, smooth (Pl. I a). 

Sucrose nitrate agar: Growth thin, pale 
olive-buff. Aerial mycelium pale vinaceous- 
fawn. No soluble pigment. 

Glucose-asparagine agar: 
ate, wrinkled. Aerial mycelium white. Solu- 


Growth moder- 


ble pigment brown. 
Calcium malate agar: Growth moderate, 


DESCRIPTION OF SPECIES OF STREPTOMYCES 247 


cream to buff. Aerial mycelium white. No 
soluble pigment. 

Nutrient agar: Growth moderate to good, 
light brown. Aerial mycelium white. Solu- 
ble pigment light brown. 

Starch agar: Growth moderate, thin; pale 
olive-buff reverse. Aerial mycelium white. 
No soluble pigment. Strong hydrolysis. 

Potato: Growth poor, waxy, wrinkled, 
brown. No aerial mycelium. Soluble pig- 
ment dark brown. 

Gelatin: Moderate surface growth. Aerial 
mycelium white. Soluble pigment dark 
brown. No liquefaction. 

Milk: Growth poor. No peptonization. 

Nitrate reduction: Negative. 

Production of HS: Variable. 

Antagonistic properties: Produces a basic 
antibiotic, netropsin. 

Remarks: Ettlinger et al. (1958) report 
this organism to be melanin-negative. They 
also consider S. cznnamomeus as closely re- 
lated. 


162. Streptomyces niger (Rossi-Doria, 
1891; emend. Krassilnikov, 1949) Waksman 
(Rossi-Doria, E. Ann. igiene, 1: 399-43 
1891; Krassilnikov, N. A. Actinomycetales. 
Izvest. Akad. Nauk. SSSR, Moskau, p. 53, 
1941). 

Morphology: Substrate growth of soft 
consistency. Aerial mycelium produced only 
on potato and synthetic agar. Sporophores 
formed only seldom; open spirals, with 3 to 
5 turns. Spores oval. 

Synthetic agar: Growth black. Aerial 
mycelium dark gray. No soluble pigment. 

Nutrient agar: Growth black. Soluble 
pigment brown. 

Gelatin: Slow liquefaction, 
Melanin-negative (?). 

Milk: No change. 

Starch: No growth. 

Cellulose: No growth. 

Nitrate reduction: Negative. 

Sucrose: No inversion. 

Temperature: Optimum 25-30°C. 


in 30 days. 


Antagonistic properties: None. 

Remarks: This is a very unstable species 
which dies out rapidly. It easily mutates, 
giving rise to colorless cultures, producing 
no aerial mycelium. It appears 
transition form, if not a true Nocardia. A. 
niger aromaticus Berestnew and A. nigrificans 
(Kriiger) Wollenweber are listed by Krassil- 
nikov as varieties of A. niger. In view of the 
formation of a soluble brown substance on 


to be a 


certain protein media, this organism may 
belong to one of the chromogenic groups. 
163. Streptomyces nigrifaciens Waksman, 
1919 (Waksman, 8. A. Soil Sci. 8: 167-168, 
1919). 
Morphology: Sporophores branching with 


tendency to curl; no true spirals. Spores 
oval-shaped to elliptical. 
Sucrose nitrate agar: Growth colorless. 


Aerial mycelium thin, gray. No soluble pig- 
ment. 

Glucose-asparagine agar: Growth cream- 
colored. Aerial mycelium mouse-gray with 
white patches. 

Nutrient agar: Growth thin, cream-col- 
ored. Aerial mycelium gray. Soluble pigment 
brown. 

Starch agar: 
yellow. Aerial mycelium light 
Hydrolysis imperfect. 

Ege media: Growth abundant, dark 
brown. No aerial mycelium. Purplish zone 
around growth. 


cream-colored to 
buff-gray. 


Growth 


Potato: Growth gray becoming dark. 
Aerial mycelium white, appearing late. 


Soluble pigment black. 

Gelatin: Growth cream-colored to brown- 
ish. Aerial mycelium white. Soluble pigment 
brown. Liquefaction slow. 

Milk: Surface growth dark brown. Aerial 
mycelium white. Coagulation and slow pep- 
tonization. 

Nitrate reduction: Positive. 

Sucrose: No inversion. 

Cellulose: No growth. 

Habitat: Pineapple soil in Hawaii. 


248 


Remarks: This organism had been de- 
scribed by Waksman (1919) as Actinomy- 
ces 145, but never named before. 

Type culture: IMRU 3067. 

164. Streptomyces nitrificans Schatz et al., 
1954 (Schatz, A., Isenberg, H. D., Angrist, 
A. A., and Schatz, V. J. Bacteriol. 68: 1-4, 
1954). 

Morphology: Sporulating hyphae straight, 
branched. 

Most solid and liquid media: Growth 
gray, with a pink to buff reverse. No solu- 
ble pigment. 

Blood agar: Growth brick-red. No hemoly- 
sis. 

Potato: Growth wrinkled. 

Nitrate reduction: Positive. 

Milk: No coagulation; slow peptonization. 

Starch: Hydrolysis. 

Gelatin: No liquefaction. 

Cellulose: Not attacked. 

Remarks: S. nitrificans grows well on a 
variety of substrates, such as ethyl carbam- 
ate. With ammonia providing nitrogen in the 
basal medium, glucose, sucrose, mannitol, 
sorbitol, glycerol, ethanol, n-propanol, ace- 
tate, lactate, succinate, fumarate, and citrate 
permitted good growth. In a glucose con- 
taining medium, ammonia, nitrite, nitrate, 
urea, and guanidine were satisfactory sources 
of nitrogen. Several amino acids, purines, 
and miscellaneous other nitrogenous com- 
pounds, supped alone or with glucose in 
the basal medium, supported growth. 

The organism grew as well on carbamate 
when first isolated from a carbamate-enrich- 
ment culture as it did after serial transfer 
over a 2-year period on various simple and 
complex media containing no carbamate. 

In addition to its apparently unique abil- 
itv to grow on carbamate as sole substrate, 
this culture also produced nitrite from car- 
bamate. It did not oxidize the carbamate 
nitrogen beyond the nitrite stage. Hirsch 
(1960) considers this organism as a Nocardia 


THE ACTINOMYCETES, Vol. II 


(N. nitrificans) capable of utilizing petro- 
leum. 


165. Streptomyces  nitrosporeus Okami, 
1952 (Okami, Y. J. Antibiotics (Japan) 5: 
477-480, 1952). 

Morphology: Aerial mycelium. straight, 
formed in clusters or tufts. Spores elliptical 
to oval. 

Sucrose nitrate agar: Substrate growth 
colorless, grayish. Aerial mycelium blackish- 
oray. 

Gelatin: Limited growth in liquefied zone. 
Liquefaction rapid. Soluble pigment yellow- 
ish-brown. 

Milk: Growth cream-colored to brownish. 
Strong coagulation and peptonization. 

Starch: Strong hydrolysis. 

Cellulose: Attacked. 

Nitrate reduction: Vigorous. 

Tyrosinase reaction: Negative. 

Production of HoS: None. 

LoefHler’s media: Growth thin. 
Aerial mycelium gray. Soluble pigment 
limited. Rapid liquefaction of serum. 

Carbon utilization: Utilizes arabinose, 
galactose, glucose, maltose, rhamnose, xy- 


serum 


lose, and glycerol; does not utilize sucrose, 
fructose, inulin, lactose, mannitol, raffinose, 
or sorbitol. 

Antagonistic properties: Produces an anti- 
biotic, nitrosporin (proactinomycin?). 

Habitat: Soil in Japan. 

Remarks: Resembles S. griseolus and S. 
cellulosae. 

Type culture: IMRU 3728; ATCC 12,769. 


166. Streptomyces niveoruber Ettlinger et 
al., 1958 ‘(Bttlinger, L., Corbaz,.-Resand 
Hitter, R. Arch. Mikrobiol. 31: 350, 1958). 

Morphology: Long, straight sporophores, 
monopodially branched, forming open, regu- 
lar spirals. Spores smooth (PI. II k). 

Glycerol nitrate agar: Growth hght yellow 
or carmine-red. Aerial mycelium sparse, 
chalk-white. 

Glucose-peptone agar: Growth white-yel- 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


low to carmine-red. Aerial mycelium abun- 
dant, white. Soluble pigment 
carmine-red. 

Calcium malate agar: Growth light yellow 
to ight carmine. No aerial mycelium. 

Starch agar: Growth light yellow-red to 
carmine-red. Aerial 
white. Limited hydrolysis. 

Potato: Growth light brown. Aerial myce- 
lium powdery, chalk-white. Melanin-nega- 
tive. 

Gelatin: Growth carmine-red. Aerial my- 
celium sparse. Trace of liquefaction. No 
soluble pigment. 

Milk: Pellicle hight yellow. Aerial myce- 
lium sparse. Coagulation limited; no pep- 
tonization. 

Antagonistic properties: 
biotic cimerubin. 

Habitat: Soils in England and Germany. 


somewhat 


mycelium abundant, 


Produces anti- 


167. Streptomyces niveus Smith et al., 1956 
(Smith, C. G., Dietz, A., Sokolski, W. T., 
and Savage, G. M. Antibiotics & Chemo- 
therapy 6: 135-142, 1956). 

Morphology: Sporophores straight at the 
base, corkscrew-coiled at tip, occur in clus- 
ters and bear oblong spores. 

Sucrose nitrate agar: Growth cream-col- 
ored. Aerial mycelium white. Soluble pig- 
ment yellow. 


Calcium malate agar: Growth cream- 
colored. Aerial mycelium white. Soluble 
pigment yellow. 

Nutrient agar: Growth cream-colored. 


Aerial mycelium trace, gray-white. Soluble 
pigment yellow. 

Gelatin: Growth good. Liquefaction par- 
tial. No soluble pigment. 


Nutrient starch agar: Growth  vellow. 
Aerial mycelium cream-pink. Hydrolysis 


good. 
Tyrosine agar: Soluble yellow pigment. 
Milk: Ring on surface; floeculent growth 
at bottom. Positive peptonization. 
Production of H.S: Negative. 
Carbon utilization: b-xylose, b-arabinose, 


249 


rhamnose, D-fructose, D-galactose, D-glucose, 
D-mannose, maltose, sucrose, lactose, cello- 
biose, raffinose, dextrin, inulin, soluble starch, 
glycerol, duleitol, bD-mannitol, b-sorbitol, im- 
ositol, salicin, sodium formate, sodium ox- 
alate, sodium tartrate, sodium acetate, so- 
dium citrate, and sodium succinate utilized. 
Phenol, cresol, and sodium salicylate not 
utilized. 

Nitrate: No reduction. 

Antagonistic properties: Produces strepto- 
nivicin, a form of novobiocin. 

Habitat: Soil. 

Remarks: According to Kuroya et al. 
(1958), this organism is related if not identi- 
cal to S. griseoflavus. 


168. Streptomyces noboritoensis Isono. et 
al., 1957 (Isono, K., Yamashita, S., Tomi- 
yama, Y., Suzuki, S., and Sakai, H. J. Anti- 
biotics (Japan) 10A: 21-30, 1957). 

Morphology: Aerial mycelium long and 
wavy; no regular spirals. 
nitrate Growth 
Aerial mycelium slight. Soluble pigment 


Sucrose agar: colorless. 
absent or pale yellow. 

Glucose-asparagine 
dark 
pale violet-gray. 
slightly brownish. 

Nutrient agar: Growth flat, pale gray, 
smooth and restricted. No aerial mycelium. 
Soluble pigment dark red-brown. 

Starch agar: Growth dry, wrinkled, pale 


agar: Growth pale 
brown. Aerial mycelium 
No soluble pigment or 


brown. to 


grayish-brown. Aerial mycelium pale-gray, 
cottony. Weak diastatic action. 

Gelatin: Growth dark brown. Soluble pig- 
ment dark brown. Liquefaction absent or 


slight. 
Potato: Growth flat, wrinkled, black. 
Aerial mycelium grayish-white in some 


strains. Color of plug black. 

Carbon utilization: Glucose, lactose, man- 
nitol, trehalose, and raffinose well utilized. 
Utilization of arabinose, inositol, salicin, 
and xylose limited. Rhamnose and sucrose 


not utilized. 


250 


Antagonistic properties: Produces anti- 
biotic homomycin-hygromycin. 

Remarks: S. noboritoensis belongs to the 
group of chromogenic actinomycetes closely 
related to S. cinnamonensis, S. flavochromo- 
genes, S. phaeochromogenes, S. aureus, and 
S. tanashiensis. They differ in spiral forma- 
tion, pigmentation on synthetic media, ni- 
trate reduction, and production of anti- 
biotics. 

169. Streptomyces nodosus Trejo, W. nov. 
Sp: 

Morphology: Aerial mycelium forms open 
and closed spirals, the latter predominating 
as tightly knotted coils. Spores spherical to 
oval, 0.5 to 1.0 by 1.0 p. 

Sucrose nitrate agar: Growth white-green- 
ish. Aerial mycelium pearl-gray to dawn- 
eray. 

Nutrient agar: Substrate growth scant. 
No aerial mycelium. No soluble pigment. 
Melanin-negative. 

Oatmeal agar: Growth black with a buff 
margin. Aerial mycelium deep olive-gray. 
Reverse: olivaceus to black with a peripheral 
ring of cream-buff to chamois. No soluble 
pigment. 

Potato: Growth buff. Aerial mycelium 
light olive-gray. No darkening of plug. 

Milk: Rapidly peptonized. 

Gelatin: Rapidly hydrolyzed. 

Nitrate reduction: Positive. 

Starch: Strong hydrolysis. 

Tyrosine: Utilized with no melanin forma- 
tion. 

Carbon utilization: Utilizes mannitol, in- 
ositol, rhamnose, xylose, D-fructose, treha- 
lose, and melibiose. Does not utilize adoni- 
tol, sorbitol, arabinose, cellulose, sucrose, 
lactose, sodium acetate, esculin, or dextrin. 

Antagonistic properties: Produces an anti- 
fungal antibiotic, amphotericin. 


* Personal communication from Squibb Insti- 
tute for Medical Research (1958). 


THE ACTINOMYCETES, Vol. II 


Source: Isolated from soil in South 
America. 

Remarks: This culture was specially de- 
scribed for this treatise. It appears to be 
closely related to S. rutgersensis. 


170. Streptomyces noursei Hazen and 
Brown, 1950 (Hazen, E. L. and Brown, R. 
Science 112: 423, 1950; Proc. Soc. Exptl. 
Biol. Med 76: 93, 1951; Science 117: 609, 
1953). 

Morphology: Sporophores produced as 
side branches of sterile aerial hyphae; occa- 
sionally produce open spirals and, according 
to Ettlinger et al. (1958), also some verticils. 
Spores round to oval, with thin long spines 
CPE ey 

Sucrose nitrate agar: Growth scanty, col- 
orless, flat. No aerial mycelium. 

Glucose-asparagine agar: Growth wrin- 
kled, tan-colored, with gray and white knob- 
like projections. Reverse of growth dark 
eray. Aerial mycelium white, then reddish- 
eray, finally ash-gray; limited shell-pink 
diffusible pigment. 

Glucose-peptone agar: Growth good, 
folded, brown. Aerial mycelium white, turn- 
ing gray. Soluble pigment brown or pome- 
eranate-purple. 

Starch agar: Growth in form of discrete 
colonies. Aerial mycelium white in center, 
periphery colorless and embedded. Hydroly- 
Sis. 

Potato: Growth folded. Aerial mycelium 
chalky white. At 35-36°C a reddish-purple 
pigment is formed. 

Gelatin: Rapid 
negative. 

Milk: Coagulation, followed by peptoni- 
zation. 

Cellulose: Growth poor. 

Nitrate: Traces of nitrite produced. 

Production of HS: Negative. 

Blood agar: Growth consists of convex, 
lobate colonies, with central perforation. 
Aerial mycelium heavy, chalky white. No 
hemolysis, but darkening of blood. 


liquefaction. Melanin- 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Antagonistic properties: Produces an 
antifungal agent, nystatin. 


Type culture: IMRU 3771. 


171. Streptomyces novaecaesareae (Waks- 
man and Curtis, 1916) Waksman and Hen- 
rici, 1948 (Waksman, 8. A. and Curtis, R. E. 
Soil Sci. 1: 111, 1916, 8: 158, 1919). 

Morphology: Aerial mycelium forms both 
straight and spiral (dextrorse) sporophores. 
Spores oval to elongate. 

Sucrose nitrate agar: Growth gray, be- 
coming bluish, glossy, much wrinkled. Aerial 
mycelium white, appears late. Soluble pur- 
ple pigment formed. 

Glucose-asparagine agar: 
stricted, gray, becoming red. 

Nutrient agar: Growth thin, cream-col- 
ored. 

Potato: Growth wrinkled, cream-colored, 
turning yellowish. Melanin-negative. 

Gelatin: Surface colonies small, cream- 
colored. Liquefaction slow. 

Milk: Gray ring. Coagulation slow; pep- 
tonization slow. 

Starch agar: Colonies restricted, circular, 
bluish-violet. Positive hydrolysis. 

Nitrate reduction: Positive. 

Production of H.S: Negative. 

Temperature: Optimum, 37°C. 

Antagonistic properties: Negative. 

Remarks: At first this organism was des- 
ignated as A. violaceus-caesari. This species 
is considered by Krassilnikov as synonymous 
with A. violaceus (Rossi-Doria) Gasperini. 
It appears to be related to S. violaceoruber. 


Growth — re- 


172. Streptomyces odorifer — (Rullman 
emend. Lachner-Sandoval, 1898) Waksman 
(Lachner-Sandoval, V. Ueber Strahlenpilze. 
Strassburg, 1898). 

Morphology: Sporophores long, straight, 
branching, forming spirals. Spores spherical. 

Sucrose nitrate agar: Growth cream-col- 
ored, with trace of brown. Aerial mycelium 
abundant, cream-colored. 

Glucose-asparagine agar: Growth cream- 


251 


colored to brownish. Aerial mycelium abun- 
dant, cream-colored. Soluble pigment faint 
brownish. 

Nutrient agar: Growth folded, brown. 
Aerial mycelium white around edge. Soluble 
pigment faint brown. 

Starch agar: 
brown. Aerial mycelium abundant, cream- 
colored to straw-colored. No soluble pig- 
ment. Hydrolysis good. 


Growth cream-colored to 


Maltose-peptone agar: Foulerton and 
Price-Jones (1902) described growth as 


“raised, drab-colored, semi-translucent, the 
surface becoming reticulated; soluble pig- 
ment deep brown; gelatin liquefied, with 
light brown pigmentation.” 

Potato: Growth folded, brownish. Aerial 
mycelium cream-colored. Soluble pigment 
faint brown. 

Gelatin: Surface ring cream-colored. Ae- 
rial mycelium thin, white. No soluble pig- 
ment. Liquefaction slow. 

Milk: Surface ring colorless to brownish. 
No aerial mycelium. No coagulation; some 
peptonization. 

Cellulose: Good growth. 

Sucrose: Inversion. 

Paraffin and fats: Good growth. 

Nitrate reduction: Positive. 

Production of HS: Negative. 

Odor: Strong, characteristic of soil. 

Antagonistic properties: Some strains give 
positive effects, others are negative. 

Habitat: Soil. 

Type culture: IMRU 3334. 


173. Streptomyces oidiosporus (Krassilni- 
kov, 1941) Waksman (Krassilnikov, N. A. 


Actinomycetales. Izvest. Akad. Nauk. 
SSSR, Moskau, p. 23, 1941). 
Morphology: Sporophores — straight or 


wavy, never forming spirals; short or long, 
frequently forming broom-shaped structures. 
Spores 1.0 to 1.8 by 0.5 to 1.0 u, frequently 
appearing as double cocci or segmented 
spores (oidiospores). 

Agar media: Growth red or rose to pale; 


252 THE ACTINOMYCETES, Vol. II 


pigment insoluble in medium. Aerial myce- 
lium poorly developed, velvety, rose-white. 
Gelatin: Aerial mycelium weakly devel- 
oped, frequently lacking; aerial hyphae 
short, rose-white. Liquefaction weak. 
Milk: No coagulation; peptonization posi- 
tive. 
Starch: Rapid hydrolysis of starch. 
Cellulose: No growth. 
Nitrate reduction: Positive. 
properties: 
positive 


None. Jolly 
effects for his 


Antagonistic 
(1956) obtained 
strain. 

Habitat: Rarely found in soil. 

Remarks: The resembles SS. 
ruber and S. longispororuber. Some strains 
were obtained as variants of Nocardia 
rubra. Jolly (1956) reported the isolation of 
a strain of S. otdiosporus from an Italian 


organism 


soil. 


174. Streptomyces  olivaceus (Waksman, 
1919) Waksman and Henrici, 1948 (Waks- 
man, S. A. Soil Sci. 8: 168, 1919). 

This organism was first described as 
strain No. 206 by Waksman (1919). It was 
used by Jensen (1930) for comparison with 
his own isolates. It was studied more re- 
cently by Shinobu (1958) and Ettlinger 
et al. (1958). 

Morphology: § Sporophores — branched 
monopodially, straight or somewhat wavy; 
no true spirals on most media; a few long, 
open spirals on calcium malate agar. Spores 
spherical and oval, 0.8 to 1.2 uw; surface 
smooth (Pl. III). 

Sucrose nitrate agar: Growth abundant, 
yellow to olive-ocher, reverse yellow to al- 
most black. Aerial mycelium ash-gray to 
light drab. 

Glucose-asparagine agar: Growth yellow 
to light olive to olive-gray. Aerial mycelium 
light olive-gray to light brownish-gray with 
greenish tinge. No soluble pigment. 

Calcium malate agar: Growth greenish- 
yellow to yellow. Aerial mycelium yellowish- 


white to yellowish-gray. Soluble pigment 
yellow. 

Nutrient agar: Growth white, glistening. 
No soluble pigment. 

Starch agar: Growth brownish-yellow to 
yellowish-green. Aerial mycelium brownish- 
white. Hydrolysis strong. 

Potato: Growth abundant, much wrin- 
kled, elevated, gray, turning sulfur-yellow 
on edge. Melanin-negative. 

Gelatin: Liquefaction rapid. No soluble 
pigment. 

Milk: Growth faint, pinkish; coagulation 
and peptonization rapid. 

Cellulose: Growth good. 

Mannase: Reaction strong, according to 
Shinobu (1958). 

Nitrate reduction: Positive. 

Production of HS: Negative. 

Tyrosinase reaction: Although this organ- 
ism has been considered as melanin-nega- 
tive, Shinobu (1958) reported a positive 
reaction. 

Temperature: Optimum 25°C. 

Carbon According to Shinobu 
(1958), S. olivaceus rapidly utilizes xylose, 
rhamnose, fructose, 


sources: 


galactose, sucrose, lac- 
tose, and mannitol; slow utilization: treha- 
lose, raffinose, and inositol. 

Antagonistic properties: Various strains 
produce a variety of antibiotics, including 
streptomycin, olivacein, and granaticin. 

Habitat: Very common in soil. 

Remarks: Krassilnikov (1949) placed the 
organism in the A. flavus group. Ettlinger 
et al. (1958) considered the following organ- 
isms as belonging to S. olzvaceus: S. felleus, 
S. flavus, S. griseolus, S. halstedii, S. nar- 
bonensis, S. scabies (sic), and S. verne. 

Type culture: IMRU 3335. 


175. Streptomyces olivochromogenes (Waks- 
man, 1919) Waksman and Henrici, 1948 
(Waksman, S. A. Actinomyces No. 205, Soil 
Sci. 8: 106, 1919). 

Morphology: Sporophores form numerous 
closed spirals. Spores oval or elliptical. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


nitrate Growth white, 
spreading. Aerial mycelium ash-gray with 
brownish tinge. No soluble pigment. 

Glycerol malate agar: Growth colorless. 
Aerial mycelium light grayish-olive to dark 
oray. 

Glucose-asparagine agar: Growth abun- 
dant, natal-brown to almost black. Aerial 
mycelium white with gray tinge. Soluble 


Sucrose agar: 


pigment brownish. 

Nutrient agar: Growth wrinkled, brown, 
becoming gray-green. Aerial mycelium 
white. Soluble pigment brown. 

Starch agar: Growth transparent, spread- 
ing. Aerial mycelium buff-gray. Rapid hy- 
drolysis. 

Potato: Colonies small, wrinkled, black. 
No aerial mycelium. Soluble pigment black. 

Gelatin: Surface growth cream-colored, 
spreading. Aerial mycelium white. Soluble 
pigment dark brown to deep olive-green. 
Slow liquefaction. 

Milk: Dark brown ring. Coagulation and 
peptonization. 

Cellulose: Growth faint. 

Nitrate reduction: Faint reduction to ni- 
trite. 

Sucrose: Invertase positive with good 
growth. 

Temperature: Optimum, 37°C. 

Antagonistic properties: Positive. 

Habitat: Soil, water, river mud. 

Remarks: Ettlinger et al. (1958) consid- 
ered this organism as a strain of S. griseus. 
KrassilInikov (1949) considered it as a vari- 
ety of A. chromogenes. 


176. Streptomyces olivoreticuli Arai et al., 
1957 (Arai, T., Nakada, T., and Suzuki, 
M. Antibiotics & Chemotherapy 7: 435-442, 
1957). 

Morphology: Sporophores form primary 
and secondary verticils; secondary may also 
be formed as tip clusters. Spores spherical 
to oval. 

Sucrose nitrate agar: Growth thin, yellow 
to brown. Aerial mycelium scant, later be- 


253 


coming cottony, white with yellowish tinge. 
Soluble pigment faint brown or absent. 

Glucose-asparagine agar: Growth thin, 
hight brown to olive-drab. Aerial mycelium 
cottony, white with faint yellow to grayish- 
pink tinge. 

Nutrient agar: Growth limited, brownish. 
Aerial mycelium grayish-white. Soluble pig- 
ment light brown. 

Gelatin: Surface growth poor. Liquefac- 
tion slow, later becoming rapid. Soluble pig- 
ment brown. 

Blood agar: Strong hemolysis. 

Potato: Growth wrinkled, dark brown. 
Aerial mycelium abundant, powdery, cream- 
colored to tea-green. Soluble pigment brown. 

Milk: Ring on surface brown. Coagulation 
with limited peptonization. Soluble pigment 
brown. 

Nitrate reduction: Negative. 

Starch: Hydrolysis. 

Cellulose: No decomposition. 

Antagonistic properties: Produces anti- 
biotic viomycin. 

177. Streptomyces  olivoverticillatus —Shi- 
nobu, 1956 (Shinobu, R. Mem. Osaka Univ. 
B (N. 8.) 5: 84-93, 1956). 

Morphology: Sucrose-ammonium agar 
most suitable for microscopic study. Pri- 
mary and secondary verticils produced, 
branches issuing sometimes closely, near the 
the sporulating hyphae, forming 
tuft-like 
spherical to elliptical, 0.6 to 0.8 u. 


top of 
cluster-like or branches. Spores 
Sucrose nitrate agar: Trace of growth. 
Glucose-asparagine agar: Growth thin, 
moderate, pale olive to pale dark yellow. 
Aerial mycelium thin, partially yellowish- 
oray. 
Nutrient 
brown. Aerial mycelium olive-gray to yellow 


agar: Growth heavy, deep 
to green. Soluble pigment brown. 
Potato: Aerial 


mycelium yellow-white to yellow-gray. Solu- 


Growth heavy, brown. 


ble pigment brown. 


Milk: Growth brown. Aerial mycelium 
scant, yellow-white. Soluble pigment brown. 

Gelatin: Liquefaction weak. 

Starch: Rapid hydrolysis. 

Tyrosinase reaction: None. 

Nitrate reduction: Negative. 

Cellulose: No growth. 

Carbon utilization: Fructose and inositol 
utilized. Xylose, rhamnose, sucrose, lactose, 
raffinose, and mannitol not utilized. 

Habitat: Soil in Japan. 


178. Streptomyces omiyaensis Umezawe 
et al., 1949 (Umezawa, H., Tazaki, T., 
Okami, Y., and Fukuyama, 8. J. Antibiotics 
(Japan) 3: 294-296, 1949). 

Morphology: Aerial mycelium 
scant branching. Sporophores straight, no 
spirals. Spores 1.0 to 1.2 by 2 to 3 u. 

Sucrose nitrate agar: Growth thin, trans- 
parent, cream-colored to dark. Aerial my- 


shows 


celium absent, or scant, white. No soluble 
pigment. 

Nutrient agar: Growth wrinkled, white to 
cream-colored. No aerial mycelium. No 
soluble pigment. Melanin-negative. 

Starch agar: Colorless thin colonies, al- 
most all submerged. No aerial mycelium. 
No soluble pigment. Hydrolysis. 


Gelatin: Growth on surface white. No 
soluble pigment. Liquefaction slight in 


crateriform. 
Potato: Growth white to cream-colored. 
No aerial mycelium. No soluble pigment. 
Milk: Growth white. Peptonization rapid. 
Acid formed. 
Antagonistic properties: Produces the 
antibiotic chloramphenicol. 
Habitat: Soil. 


Remarks: Related to S. cacaoz. 


179. Streptomyces orientalis Pittenger and 
Brigham, 1956 (Pittenger, R. C. and Brig- 
ham, R. B. Antibiotics & Chemotherapy 6: 
642-647, 1956). 

Morphology: Substrate growth made up 
of typical prostrate, much-branched myce- 


THE ACTINOMYCETES, Vol. II 


lium. Aerial mycelium abundant if starch is 
used as carbon source. Straight or irregularly 
branched sporophores made up of cylindrical 
to ovoid spores, 0.7 to 1.0 by 1.4 to 1.8 up. 

Sucrose nitrate agar: Growth secant to 
moderate, pale cream color. Aerial mycelium 
trace of off-white. No pigment or pale yel- 
lowish-brown to light brown soluble pigment 
may be formed. 

Glucose-asparagine agar: Growth moder- 
ate to good, cream-colored. Aerial mycelium 
pale to cream-colored, powdery. Soluble pig- 
ment pale greenish-yellow. 

Glycerol malate agar: Growth pale cream 
to intense cream-yellow. Aerial mycelium 
whitish in color. No soluble pigment. Insolu- 
ble malate cleared in agar around growth. 

Nutrient agar: Growth cream-colored. Ae- 
rial mycelium whitish. No soluble pigment. 

Starch agar: Growth moderate, cream-col- 
ored to buff to brown. Aerial mycelium 
white, becoming pale cream and _ finally 
grayish. Soluble pigment cream-yellow, be- 
coming pale brown. Hydrolysis limited. 

Potato plug: Growth shows slightly rough 
surface. Aerial mycelium white. Slight to 
moderate amount of brown discoloration of 
plug. 

Gelatin: Growth flocculent, not forming 
intact pellicle. Aerial mycelium scant, white. 
No soluble pigment. Liquefaction moderate. 

Milk: Heavy wrinkled pellicle, with dull 
gray aerial mycelium. No coagulation. Pep- 
tonization begins in 11 to 14 days and is 
complete in 14 to 21 days. Very dark soluble 
pigment obscures litmus color. 

Cellulose: Growth good. 

Antagonistic properties: Antibiotic vanco- 
mycin produced. 

temarks: S. orientalis is most closely re- 
lated to species intermediate between S. 
albus and S. flavus, such as S. alboflavus, S. 


fllobisporus, and S. longisporoflavus. S. albo- 


gavus cannot utilize cellulose, hydrolyzes 
gelatin far less effectively than S. orzentalis, 
but attacks starch readily. Milk is feebly 


DESCRIPTION OF SPECIES OF STREPTOMYCES 2595 


digested by S. alboflavus but rapidly hy- 
drolyzed by S. orzentalis. Production of ae- 
rial mycelium by the two cultures differs on 
several media. 


180. Streptomyces  paraguayensis — (Al- 
meida, 1940) nov. comb. (Almeida, F. 
Mycopathologia 2: 201-203, 1940). 

Morphology: Thin, ramified mycelial fila- 
ments; aerial mycelium consists of thicker 
and darker filaments, | u in diameter. Gram- 
positive and nonacid-nonalcohol resistant. 

Glucose-peptone agar: Growth hard, ad- 
hering to the medium; white with dark 
center, gradually changing to dark yellow to 
almost chocolate. 

Nutrient agar: Growth rough, adhering to 
the medium; dark gray in color. 

Potato: Colonies  cerebriform, 
growth dry and friable. 

Gelatin: Growth on surface. No liquefac- 


white; 


tion. 

Milk: Surface membrane, the milk colored 
pink; no peptonization. 

Habitat: Thoracic mycetoma; dark heavy 


erains. 


181. Streptomyces parvullus Waksman and 
Gregory, 1954 (Waksman, S. A. and Greg- 
ory, F. J. Antibiotics & Chemotherapy 4: 
1050-1056, 1954). 

Morphology: Sporophores long, 
podially branched, twisting into long closed 
spirals. Spores spherical, smooth (Pl. I g). 

Sucrose nitrate agar: Growth abundant 


mono- 


with yellow reverse. Aerial mycelium ash- 
eray. Soluble pigment yellow. 

Glucose-asparagine agar: Growth yellow. 
Aerial mycelium abundant, gray. Soluble 
pigment yellow. 

Nutrient agar: Growth yellowish, covered 
with thin white aerial mycelium. Soluble pig- 
ment yellow. Melanin-negative. 

Potato: Growth orange-colored, covered 
with white to gray aerial mycelium. No solu- 
ble pigment. 

Gelatin: with 


Surface pellicle covered 


heavy gray aerial mycelium. Liquefaction 
slow. Soluble pigment yellow. 

Milk: Surface growth heavy, greenish-yel- 
low. Aerial mycelium abundant, gray. Solu- 
ble pigment brown. No coagulation, very 
slow peptonization. 

Production of HoS: Negative. 

Antagonistic properties: Produces actino- 
mycin D. 

Habitat: Soil. 

Type culture: IMRU 3677. 

182. Streptomyces parvus (Xrainsky, 1914) 
Waksman and Henrici, 1948 (Krainsky, A. 
Centr. Bakteriol. Parasitenk. Abt. II., 41: 
685-686, 1914). 

Morphology: 
branched, or wavy; no true spirals; some 
strains, however, produce spirals. Spores 
oval, 0:9. to L.d:by 1.2 to-1-8 pu: 

Sucrose nitrate agar: Growth yellow, rose 
or red. Aerial mycelium light yellow to 
white-rose. Soluble pigment rose-colored to 


Sporophores straight, 


bright yellow. 

Calcium malate agar: Small yellow colo- 
nies. Light yellow aerial mycelium. 

Nutrient agar: Growth yellow. Aerial my- 
celum light yellow. Soluble pigment bright 
vellow. 

Potato: Growth yellow to brown-yellow. 
Aerial mycelium white to yellow. Melanin- 
negative. 

Gelatin: Growth yellow. Soluble pigment 
bright yellow. Liquefaction slow. 

Milk: No coagulation; rapid peptoniza- 
tion. 

Starch agar: Growth rose-colored. Aerial 
mycelium light gray. Hydrolysis positive. 

Cellulose: Growth good, rose-colored. Ae- 
rial mycelium yellowish-gray. 

Nitrate reduction: Weak. 

Production of H.S: Negative. 

Antagonistic properties: Produces actino- 
mycin. 

Habitat: Soil. 

Remarks: Ettlinger et al. 


(1958) con- 


sidered S. parvus as belonging to the S. 
griseus series. According to Gause ef al. 
(1957), S. parvus is a member of the series 
Fradiae. 

Type culture: IMRU 3686. 

183. Streptomyces pelletiert (Laveran, 1906) 
nov. comb. (Laveran, 8. Compt. rend. soc. 
biol. 61: 340, 1906). 

Morphology: Growth red, smooth, con- 
sisting of small, dense, pink colonies. Myce- 
lium nonsegmented, branched; hyphae 
slender, straight, and not very long. Aerial 
hyphae few, straight. 

Glucose-asparagine agar: Growth in form 
of small, hard, red or purple adherent col- 
onies. No soluble pigment. 

Glucose agar: Growth poor, in form of 
minute, pink colonies. 

Glycerol agar: Growth poor, as few moist, 
pink colonies. 

Nutrient agar: Colonies minute, colorless, 
piled up into pale pink masses. 

Potato: Growth sparse, yellowish-pink, 
irregularly piled up; later, abundant, small, 
rounded, pink Aerial mycelium 
scant, white. 

Blood agar: Colonies at first a few pin- 
head, cream-colored; no hemolysis. Later, 
dense, button-shaped, with 
narrow, fringed margin. 

Dorset’s egg medium: Growth abundant, 
wrinkled, pink skin with small discrete 
colonies at margin; later, surface rough, 


masses. 


colonies are 


mealy, with considerable liquefaction. 

Gelatin: Few pink flakes. At first slow, 
later almost complete liquefaction. 

Milk: Soft curd; gradual peptonization. 

Starch: No hydrolysis. 

Production of H.S: Negative. 

Source: Mycetoma in Nigeria. 

Remarks: In the original description of 
this culture by Laveran, the organism was 
called Micrococcus pelletiert, because no my- 
celium was seen, only coccoid bodies. N. 
indica was regarded as identical by Pinoy. 
N. genesti Froes was described as closely 


THE ACTINOMYCETES, Vol. II 


allied (Erikson, 1935); the distinction was 
founded upon the fact that the red grains 
were smaller and much more numerous. A. 
africanus is considered as a synonym of this 
organism. According to Mariat (1958), S. 
pelletiert hydrolyzes gelatin, serum albumin, 
casein, and egg albumin; it utilizes urea but 
not (NH4).SO, and KNO; as_ nitrogen 
sources; it does not utilize xylose, galactose, 
maltose, starch, mannitol, or paraffin as car- 
bon sources. The species S. africanus is 
indistinguishable from S. pelletier?. 


184. Streptomyces pentaticus Umezawa 
and Tanaka, 1958 (Umezawa, 8. and 
Tanaka, Y. J. Antibiotics (Japan) 11A: 26- 
29, 1958). 

Morphology: Straight sporophores pro- 
duce primary and secondary verticils. Spores 
can scarcely be observed. 

Sucrose nitrate agar: Growth poor, trans- 
parent, penetrates deeply into medium. No 
aerial mycelium. No soluble pigment. 

Glucose-asparagine agar: Growth 
less, becoming purplish-pink to dull red- 
purple, deep into medium. Aerial mycelium 
white, sometimes pink. Soluble pigment 
faint brown. 

Calcium malate agar: Growth red, irreg- 


color- 


ular margin. No aerial mycelium. Soluble 
pigment faint brown. 

Nutrient agar: Growth wet, colorless or 
brownish-white. No aerial mycelium. Solu- 
ble pigment brown. 

Starch Growth 
yellow, penetrates deeply into medium. Ae- 
rial mycelium white, partially pinkish cot- 


agar: colorless or pale 


tony colonies. No soluble pigment. Starch 
hydrolyzed. 

Gelatin: Growth consists of reddish colo- 
nies produced on surface. No aerial myce- 
lium. Soluble pigment deep brown. Rapid 
liquefaction. 

Potato: Growth wrinkled, wet, grayish- 
brown. No aerial mycelium. Soluble pig- 
ment brownish-black. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Milk: Surface ring dull yellow. Coagula- 
tion and peptonization. 

Antagonistic properties: Produces an anti- 
fungal polyenic antibiotic, pentamycin. 

Habitat: Soil in Japan. 

Remarks: Resembles  S. 
which differs from the strain producing 


rubrireticuli, 


pentamycin in the following ways: spirals 
are formed; growth on nutrient agar is red; 
growth on milk is abundant and red; cellu- 
lose and sucrose are utilized. 


185. Streptomyces phaeochromogenes 
(Conn, 1917) Waksman and Henrici, 1948 
(Gonn: HH. J. (N. Y: Agr.. Expt: Sta. Tech. 
Bulle 60.1917). 

This culture has been studied by Conn 
(1917), Waksman (1919), Jensen (1931), 
Krassilnikov (1949), Kutzner (1956), and 
Ettlinger et al. (1958). 

Morphology: Sporophores form narrow, 
open, elongated, sinistrorse spirals (Conn, 
Waksman, Jensen, Krassilnikov). Kutzner 
(1956) examined 25 strains belonging to this 
species; only five of them produced spirals. 
Ettlnger et al. (1958) could not find any 
spirals on any of the strains obtained from 
various culture collections. Spores spherical 
to short rods; surface smooth (Pl. I d). 

Sucrose nitrate agar: Growth brown to 
almost black. Aerial mycelium abundant, 
white with brownish shade. Soluble pigment 
brown to dark brown. 
buff to 
brown. Aerial mycelium white. Soluble pig- 


Calcium malate agar: Growth 
ment brown. 
Nutrient 


later turning nearly black. Aerial mycelium 


agar: Growth gray to brown, 
white to gray, often absent. Soluble pigment 
deep red-brown. 

Starch agar: Growth brown. Hydrolysis 
medium. 

Potato: Growth brown to almost black. 
No aerial mycelium. Soluble pigment dark 
brown to black. 

Gelatin: Surface 


growth abundant, 


spreading, cream-colored, becoming brown. 

Liquefaction slow. Soluble pigment brown. 
Milk: Dark, almost black ring: 

tion with slow peptonization. 
Nitrate: Reduction limited. 
Production of H.S: Positive. 
Temperature: Optimum 25°C. 
Antagonistic properties: Strong. 
Habitat: Soil. 
Type culture: IMRU 3338. 


coagula- 


186. Streptomyces phaeopurpureus  Shi- 
nobu, 1957 (Shinobu, R. Mem. Osaka Univ., 
B. Nat. Sci. 6: 638-67, 1957). 

Morphology: Substrate mycelium mono- 
podial, 0.4 to 0.6 uw in diameter; no frag- 
mentation. Aerial mycelium straight, usu- 
ally short. Spores spherical to elliptical, 0.6 
to 0.8 uw; rarely 1 yu. 

Sucrose nitrate agar: Growth good, brown 
to dark red. No aerial mycelium. Soluble 
pigment brown. 

Glycerol malate 
orange to purple. Aerial mycelium powdery, 
vellowish-gray to pinkish-gray. Soluble pig- 
ment red-purple to brown-purple. 

Glucose-asparagine agar: Growth moder- 


agar: Growth good, 


ate, orange to red-brown. Aerial mycelium 
moderate, in patches, pinkish-gray. Soluble 
pigment reddish-orange to reddish-brown. 

Nutrient agar: Growth good, deep brown. 
No aerial mycelium. Soluble pigment brown 
to deep reddish-brown. 

Potato plug: Growth wrinkled, reddish to 
yellowish-brown. Aerial mycelium absent or 
scant, light brownish-gray. Plug colored 
brown. 

Milk: Growth in form of deep brown ring. 
Soluble pigment brown. No coagulation; 
peptonization uncertain. 

Tyrosinase reaction: Positive. 

Gelatin: Liquefaction fairly strong. 

Diastase: Weak. 

Cellulose: Negative. 

Nitrate reduction: Negative. 

Carbon sources: Utilizes xylose, rhamnose, 


258 THE ACTINOMYCETES, Vol. II 


fructose, sucrose, lactose, raffnose, mannitol, 
and inositol. 
Habitat: Soil in Japan. 


187. Streptomyces phaeoviridis Shinobu, 
1957 (Shinobu, R. Mem. Osaka Univ., B. 
Nat. Sci. 6: 67-70, 1957). 

Morphology: Growth monopodial, hyphae 
0.4 to 0.6 uw in diameter, no fragmentation. 
Aerial mycelium short; monopodial branch- 
ing; some spirals, sinistrorse, 1 to 8 turns. 
Spores elliptical, 0.6 to 0.8 uw. 

Sucrose nitrate agar: Growth pale yellow 
to dark brown. Aerial mycelium scant, 
brownish-white. Soluble pigment yellow to 
brown to dark blue. 

Malate-glycerol agar: Growth yellowish- 
brown to dark brown. Aerial mycelium scant, 
white to brownish-white. Soluble pigment 
brown to dark brown with blue tinge. 

Glucose-asparagine agar: Growth thin 
brown to yellow-orange; aerial mycelium 
scant, white to brownish-white. Soluble 
pigment pale brown to yellow-orange. 

Nutrient agar: Growth thin, yellow-orange 
to brown. No aerial mycelium. Soluble pig- 
ment brown to dark red with purple tinge. 

Potato: Growth poor, pale brown. No 
aerial mycelium. Soluble pigment uncertain, 
probably pale brown. 

Milk: Growth good, pale yellow, partially 
blue, sometimes grayish-green. Soluble pig- 
ment absent or pale orange. Coagulation 
and peptonization. 

Tyrosinase reaction: Negative. 

Gelatin: Liquefaction variable. 

Diastase: Strong. 

Nitrate reduction: Negative. 

Cellulose: No growth. 

Carbon sources: Utilizes xylose, rhamnose, 
sucrose, fructose, raffinose, and mannitol; 
lactose and inositol uncertain. 

Habitat: Soil in Japan. 


I88. Streptomyces pilosus Ettlinger et al., 
1958 (Ettlinger, L., Corbaz, R., and Hitter, 
R. Arch. Mikrobiol. 31: 347, 1958). 


Morphology: Sporophores monopodially 
branched, with long, regular, open spirals. 
Spores covered with fine long hair. 

Glycerol nitrate agar: Growth yellow to 
yellow-brown. Aerial mycelium powdery, 
chalk-white to gray-blue. 

Glucose-asparagine agar: Growth light 
yellow. Aerial mycelium powdery, white to 
ash-gray. 

Calcium malate agar: Growth white-yel- 
low to yellow-brown. Aerial mycelium white- 
yellow to white-gray. 

Starch agar: Growth yellow-brown to red- 
yellow. Aerial mycelium scant, white to 
ash-gray. Limited hydrolysis. 

Gelatin: Growth yellow-brown. Aerial 
mycelium powdery, chalk-white. Liquefac- 
tion slow. Soluble pigment dark brown. 

Potato: Growth golden yellow. Aerial 
mycelium ash-gray. Soluble pigment dark 
brown. 

Milk: Surface growth white-gray to gray- 
ish-blue. Aerial mycelium ash-gray. No co- 
agulation; peptonization weak. 

Antagonistic properties: Positive. 

Habitat: Soil from Rome, Italy. 


189. Streptomyces platensis Pittenger and 
Gottlieb, 1954 (Pittenger, R. C. and Gott- 
heb, D. Brit. Pat. 713,795, August’ 18, 
1954*). 

Morphology: Aerial mycelium forms loose 
to tight spirals on its sporophores. Spores 
ovoid, 0.7 to 0.9 by 0.8 to 1.2 yu. 

Sucrose nitrate agar: Substrate growth 
deep olive, reverse becoming dark olive. 
Aerial mycelium pale smoke-gray with tufts 
of white; areas of black pigmented aerial 
growth may also be found, giving effect of 
a mosaic. 

Glucose - asparagine agar: Substrate 
erowth ochraceous-buff becoming tawny. 
Aerial mycelium white becoming grayish- 
olive to almost black. Soluble pigment ab- 
sent or shght, brown. 


* Supplemented by personal communication. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 259 


Calcium malate agar: Growth ochraceous- 
salmon, becoming cinnamon-buff. Aerial 
mycelium quaker-drab with areas of black 
and white. Soluble pigment slight, greenish- 
yellow. 

Nutrient agar: Poor substrate growth, 
cream-yellow becoming buff to drab. No 
aerial mycelium. Slight soluble brown pig- 


ment. 
Starch agar: Growth cream- to buff-col- 
ored. Aerial mycelium white, becoming 


mouse-gray with patches of black. Slow 
hydrolysis. 

Potato: Excellent growth. Aerial myce- 
lium white to pale mouse-gray. Soluble pig- 
ment brown. 

Gelatin: Very slow liquefaction. Melanin- 
negative. 

Milk: Growth secant, forming partial ring 
at surface. No coagulation or peptonization. 

Blood: Hemolysis. 

Cellulose: Growth slight. Aerial mycelium 
gray to black. 

Nitrate: Reduction to nitrite, especially 
with starch as source of carbon. 

Carbon utilization: Starch, malic acid, 
inositol, sodium succinate, sodium citrate, 
sorbitol, mannitol, maltose, arabinose, lac- 
tose, galactose, fructose well utilized. Dul- 
citol, raffinose, cellulose, sodium formate, 


sodium tartrate, xylose poorly utilized. 
Asparagine, rhamnose, o0-cresol, m-cresol, 


sodium acetate, inulin, sodium. salicylate 
not utilized. 

Antagonistic properties: Produces oxytet- 
racycline. 

Remarks: Tresner and Backus (1956) con- 
sidered this organism as a variant of S. 
hygroscopicus rather than a separate species. 
Ettlinger et al. (1958) came to similar con- 
clusions. 


190. Streptomyces pluricolor (Berestnew, 
1897 emend. Krassilnikov, 1941) Waksman 
(Krassilnikov, N. <A. Actinomycetales. 
Izvest. Akad. Nauk. SSSR, Moskau, p. 17, 
1949). 


Morphology: Sporophores produce nu- 
merous spirals, with 3 to 5 turns (sinistrorse). 
Spores oval, 0.9 by 0.7 u. 

Synthetic agar: Growth at first pigmented 
vellow-red, later becoming blue to blue- 
green. Aerial mycelium white-gray. The blue 
pigment dissolves into the medium. 


Nutrient agar: Soluble pigment greenish, 
fluorescent. 
Potato: Growth and soluble pigment 


sharp blue. 

Gelatin: Liquefaction rapid. 

Milk: Peptonization positive; no coagula- 
tion. 

Starch: Hydrolysis. 

Cellulose: No growth. 

Nutrient broth: Soluble pigment green, 
fluorescent. 

Sucrose: Inversion. 

Antagonistic properties: None. 

Habitat: Soil. 

temarks: Closely related to S. violace- 
oruber. A. pluricolor diffundens Berestnew is 
considered by Krassilnikov as a synonym. 

191. Streptomyces pluricolorescens Okami, 
Y. and Umezawa, H.* n. sp. 

Morphology: Aerial hyphae not flexuous, 
few branches; no spirals. 

Glycerol nitrate agar: Growth at first 
and yellowish, then yellowish- 
brown with reddish tone. Aerial mycelium 
white to olive or pinkish. Soluble pigment. 
shghtly yellowish-brown or light wine-color 
with aging. 

Glucose—asparagine agar: Growth at first 
and yellowish, then yellowish- 
brown with reddish tone. Aerial mycelium 
white to olive or pinkish. Soluble reddish- 
purple pigment occasionally produced. 

Calcium malate agar: Same as on glycerol 
nitrate agar. 


colorless 


colorless 


|Q70 


Nutrient agar: 37°C. Colorless or slight 
vellowish-brown growth with fine wrinkles. 
Aerial mycelium white. Soluble pigment ab- 
sent at first, later a brown pigment appears. 


* Personal communication from Okami. 


260 


Potato plug: Growth colorless, then 
slightly yellowish or brownish. Aerial myce- 
lium white to olive-colored. Color of plug 
unchanged. Melanin-negative. 

Gelatin: 18-20°C. Growth colorless. No 
aerial mycelium. Soluble pigment. slightly 
yellowish-brown. Gelatin liquefied. 

Milk: 37°C. Colorless to slight yellowish 
growth. No aerial mycelium. Coagulation 
with acid reaction, then peptonization. 

Starch: Hydrolysis weak to medium. 

Carbon utilization: Good growth with 
arabinose, dextrin, fructose, galactose, glu- 
cose, glycerol, maltose, mannitol, mannose, 
raffinose, rhamnose, salicin, sorbose, starch, 
sucrose, xylose, and sodium succinate. Scant 
growth with esculin, inositol, lactose, sor- 
bitol, sodium acetate, and citrate. 

Antagonistic properties: Produces anti- 
tumor substances pluramycin A and B 
(Maeda et al., 1956). 

temarks: This culture is said to be re- 
lated to S. vinaceus, but it does not produce 
blue-red pigment in reverse of growth on 
nutrient agar. 


192. Streptomyces poolensis (Taubenhaus, 
1918) Waksman (Taubenhaus, J. J. J. Agr. 
Xesearch 13: 446, 1918). 

Morphelogy: Sporophores straight. Spores 
oval to elliptical. 

Sucrose nitrate agar: Growth thin, color- 
less. Aerial mycelium white to gray. 

Glucose-asparagine agar: Growth abun- 
dant, glossy, light brown. 

Nutrient agar: Growth translucent, yel- 
lowish to brown. Soluble pigment brown. 

Potato: Growth thin, reddish-brown. Sol- 
uble pigment purplish. 

Gelatin: Liquefaction, with small, brown- 
ish flakes in fluid. 

Milk: Brownish ring. Coagulation and 
peptonization. 

Starch: Growth restricted, cream-colored. 
No hydrolysis. 

Nitrate reduction: Positive. 

Antagonistic properties: Positive. 


THE ACTINOMYCETES, Vol. II 


Habitat: Sweet-potato disease known as 
pOxs ” 


193. Streptomyces praecox (Millard and 
Burr, 1926) Waksman and Henrici, 1948 
(Millard, W. A. and Burr, 8. Ann. Appl. 
Biol. 13: 580, 1926). 

Morphology: Sporophores produce short, 
open spirals. Spores spherical or oval, 0.8 
uw in diameter. 

Sucrose nitrate agar: Growth thin, color- 
less. Aerial mycelium gray to olive-buff. On 
continued cultivation, aerial mycelium tends 
to become white. 

Nutrient agar: 
mycelium white. 

Starch media: Growth thin, cream-col- 
ored. Aerial mycelium white with greenish 
tinge. Hydrolysis positive. 

Potato: Growth lichenoid, cream-colored 
to light brown. Aerial mycelium white to 
olive-buff. Soluble pigment olive-buff to 
drab. On continued cultivation, no soluble 
pigment produced. 

Gelatin: Growth good. Aerial mycelium 
white. Liquefaction medium. Melanin-nega- 
tive. 

Milk: Surface growth cream-colored, in 


Growth colorless. Aerial 


form of ring. Aerial mycelium white. Coagu- 
lation slow; peptonization rapid. 

Nitrate: Reduction variable. 

Cellulose: Good growth, colorless. Aerial 
mycelium dark gray. 

Tyrosinase reaction: Negative. 

Production of H.S: Negative. 

Temperature: Grows well at 37.5°C 

Odor: Very strong. 

Antagonistic properties: Represses growth 
of S. scabies. 

Habitat: IKnoblike scab of potatoes. 

temarks: According to Ettlinger et al. 
(1958), this organism belongs to the S. 
griseus series. Hoffmann (1958) described a 
culture of S. praecox that produced a light 
to dark gray aerial mycelium and many 
spirals; nonchromogenic. 

Type culture: IMRU 3374. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


194. Streptomyces praefecundus (Millard 
and Burr, 1926) Waksman (Millard, W. A. 
and Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight, fre- 
quently forming brushes. Spores spherical 
to oval, 0.8 by 0.85 wu. 

Sucrose nitrate agar: Growth good, cream- 
colored. Aerial mycelium cottony, olive- 
buff. Soluble pigment cream-colored. 

Nutrient potato agar: Growth lichenoid, 
gray. Aerial mycelium smooth, white to 
yellowish. Soluble pigment golden brown. 

Potato: Growth good, wrinkled. Aerial 
mycelium white to yellowish to olive-buff. 
Soluble pigment gray to brown. 

Gelatin: Surface growth good. Aerial my- 
celium white. Soluble pigment light pink to 
dark golden brown. Liquefaction rapid. 

Milk: Surface growth good. Aerial myce- 
lium scant, white. Coagulation and peptoni- 
zation. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Temperature: Grows well at 37.5°C. 

Habitat: Potato scab and soil. 


195. Streptomyces prasinophilus Ettlinger 
et al., 1958 (Ettlinger, L., Corbaz, R., and 
Hiitter, R. Arch. Mikrobiol. 31: 345, 1958). 

Morphology: Sporophores monopodially 
branched, long, straight, with open spirals, 
usually 1 to 8 coils. Spores covered with 
long, fine hair (Pl. II, Ix). 

Glycerol nitrate agar: Growth red or red- 
brown. Aerial mycelium leek-green 
white spots. Soluble pigment pink. 

Glucose-asparagine agar: Growth  brick- 
red. Aerial mycelium white to leek-green. 
Soluble pigment brick-red. 

Glycerol malate agar: Growth brick-red. 
Aerial mycelium leek-green. Soluble pig- 
ment pink. 

Glucose-peptone agar: Growth brick-red. 

Starch-KNO; agar: Growth pink. Aerial 
mycelium white to leek-green. Soluble pig- 
ment light pink. Good hydrolysis. 

Gelatin: Bottom flakes red to yellowish, 


with 


261 


later brick-red. Slow liquefaction. No soluble 
pigment. Melanin-negative. 

Potato: Growth slow, flesh-red. No solu- 
ble pigment. 

Milk: Strong coagulation and peptoniza- 
tion. 

Antagonistic properties: Weak activity 
against gram-positive bacteria. 

Habitat: Soil in Mallorca, Spain. 


196. Streptomyces prasinus Ettlinger et al., 
1958 (Ettlinger, L., Corbaz, R., and Hitter, 
R. Arch. Mikrobiol. 31: 343, 1958). 

Morphology: Sporophores monopodially 
branched, long, straight, with open spirals, 
forming | te 2 coils. Spores covered with 
short spines (PI. IT, Ix). 

Glycerol nitrate agar: Growth colorless. 


Aerial mycelium grass-green, later dark 
green. 


Glucose-asparagine agar: Growth whitish- 
vellow. 

Glycerol malate agar: Growth copper-red. 
Aerial mycelium velvety, leek-green. 

Gelatin: Growth limited, whitish-yellow. 
No soluble pigment. Liquefaction positive. 


Starch agar: Growth reddish-brown. 
Aerial mycelium leek-green. Strong hy- 
drolysis. 

Potato: Growth limited, lght brown. 


Aerial mycelium leek-green. Melanin-nega- 
tive. 

Milk: Heavy pellicle. Aerial mycelium 
whitish-gray to greenish-gray. No coagula- 
tion; slow peptonization. 

Antagonistic properties: None. 

Habitat: Soils in Mallorea and Belgian 
Congo. 


197. Streptomyces pseudogriseolus Okami 
et al., 1955 (Okami, Y., Utahara, R., Oyagi, 
H., Nakamura, S., and Umezawa, H. J. 
Antibiotics (Japan) 8A: 126-131, 1955). 

Morphology: Sporophores produce nu- 
merous closed spirals. Spores oval to cylin- 
drical, 0.8 to 1:2 by 1:0 to: 1:5 py. 


Glycerol nitrate agar: Growth colorless to 


262 


grayish-buff. Aerial mycelium grayish-buff, 
powdery. No soluble pigment. 

Nutrient agar: Growth colorless. Aerial 
mycelium white, thin. Soluble pigment ab- 
sent or shght, brown. 

Potato: Growth colorless to slightly yel- 
lowish, elevated, wrinkled. Aerial mycelium 
white, cottony to velvety. No soluble pig- 
ment. 

Milk: Surface growth orange. Aerial my- 
celium velvety, white. Coagulation and pep- 
tonization completed in 25 to 30 days. 

Gelatin: Growth yellowish-brown. Aerial 
mycelium white to grayish. Soluble pigment 
brownish. Liquefaction weak to medium. 
Melanin-negative. 

Starch: Hydrolysis strong. 

Carbon utilization: Utilizes arabinose, 
dextrin, 7zso-dulcitol, fructose, galactose, 
glucose, glycerol, inositol, lactose, maltose, 
mannitol, mannose, rhamnose, — salicin, 
starch, sucrose, sodium acetate, sodium 
citrate, and sodium succinate. Does not 
utilize esculin, raffinose, sorbitol, or sorbose 
(inulin). 

Antagonistic properties: Produces xantho- 
mycin-like substance. 

Habitat: Isolated from soil in Japan. 

Remarks: This culture resembles S. gr7- 
seolus, but it differs in spiral formation and 
growth on potato and milk media. These 
differences may not be enough to warrant 
establishing a new species, but the produc- 
tion of xanthomycin is a property not found 
in the culture liquid of S. griseolus. 

Type culture: ATCC 12,770. 

198. Streptomyces purpureefuscus Yama- 
guchi and Saburi, 1955 (Yamaguchi, T. and 
Saburi, Y. J. Gen. Appl. Microbiol. 1: 201- 
235, 1955). 

Morphology: Aerial hyphae and 
straight; on synthetic and starch agars, they 


long 


show a tuft-forming tendency at the margin. 
Spirals not produced. Spores cylindrical, 
1.1 to 2.2 by 0.7 to 1.1 py. 


Sucrose nitrate agar: Growth thin, color- 


THE ACTINOMYCETES, Vol. II 


less, later becoming purple to dark purple; 
this property may be lost after repeated 
transter, in which case growth remains white 
to hght purple. Aerial mycelium powdery, 
white, later smoke-gray. Soluble pigment 
faint purple. 

Glycerol malate agar: Growth brownish 
to purplish-brown. Aerial mycelium white, 
smoke-gray to light grayish-olive. Soluble 
pigment light brownish-purple. 

Nutrient agar: Growth wrinkled, colorless. 
Aerial mycelium absent, or scant, white. 
Soluble pigment changes from reddish-brown 
to dark vinaceous-brown. 

Starch agar: Growth yellowish-brown to 
dark olive-buff; sometimes with hygroscopic, 
black patches. Aerial mycelium velvety, at 
first white, later olive-gray. Usually no sol- 
uble pigment is produced, but sometimes 
faint pinkish-purple is seen. Strong hy- 
drolysis. 

Potato: Growth vigorous, finely wrinkled, 
at first purplish-brown or yellowish-brown, 
later becoming black. Aerial mycelum abun- 
dant, powdery, grayish. Soluble pigment 
purplish. 

Gelatin: Growth dark brownish-gray. 
Aerial mycelium coarse, powdery, grayish- 
white. Soluble pigments yellowish-brown to 
brown and a more diffusible yellowish-green. 
Strong liquefaction. 

Milk: Growth at first dull reddish-brown, 
later purplish-brown. Soluble pigment faint 
purplish and more diffusible faint yellowish. 
Coagulation and peptonization. 

Cellulose: No growth. 

Carbon utilization: pD-xylose, L-arabinose, 
b-galactose, sucrose, lactose, raffinose, so- 
dium succinate readily utilized. L-rhamnose, 
inulin, mannitol, sorbitol, inositol, acetate, 
and citrate not utilized. 

Antagonistic properties: Active against 
gram-positive and acid-fast bacteria; pos- 
sesses antitrichomonal activity. 

Remarks: Related to S. 
cylindrosporus, and S. purpureochromogenes. 


vinaceus, S. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


199. Streptomyces purpurascens  Linden- 
bein, 1952 (Lindenbein, W. Arch. Mikrobiol. 
17: 361-383, 1952). 

Morphology: Sporophores long, straight, 
with open and closed spirals, 2 to 5 turns as 
side branches. Spores covered with long 
spines (Pl. Ib). A detailed electron micro- 
scope study of this organism has been made 
by Petras (1959). 

Glycerol nitrate agar: 
red to purple. Aerial mycelium cottony, 
white to purplish. Soluble pigment brown- 


Growth carmine- 


red. 

Glucose-asparagine agar: Growth car- 
mine-red to purple. Aerial mycelium white 
to pinkish. Soluble pigment orange to car- 
mine-red. 

Glycerol malate agar: Growth carmine- 
red. Aerial mycelium white. Soluble pigment 
orange to brick-red. 

Nutrient agar: Growth light brown, with 
dark brown reverse. Aerial mycelium white. 
Soluble pigment dark brown. Melanin- 
positive. 

Glucose-peptone agar: Growth lichenoid, 
red to red-brown. Aerial mycelium white. 
Soluble pigment light brown. 

Starch media: Growth light carmine to 
vellow-red. Aerial mycelium white. No sol- 
uble pigment. Hydrolysis strong. 

Potato: Growth reddish. 
Aerial mycelium white to gray. No soluble 
pigment. (Kutzner (1956) observed on six 
strains a gray to black pigment on potato 
plug.) 

Gelatin: Growth light brown. Aerial my- 
celium white. Soluble pigment red-brown. 
Liquefaction medium. 

Milk: Growth red to dark brown. Aerial 
mycelium white. No proteolysis. 

Cellulose: Growth white to red. 

Production of HoS: Positive. 

Antagonistic properties: Produces rhodo- 
mycin. 

Remarks: On continued growth on syn- 
thetic media, the culture may the 


brownish to 


lose 


263 


property to produce the typical pigment. It 
can be regained, however, by growth on 
organic media. This organism is considered 
by Corbaz et al. (1957) as a synonym of S. 
bobiliae, except that the latter lost the prop- 
erty of producing aerial mycelium or spores. 
Lindenbein (1952) and Frommer (1959) ob- 
tained colorless mutants from S. purpuras- 
cens. 


Type culture: IMRU 3660. 


200. Streptomyces purpureochromogenes 
(Waksman and Curtis, 1916) Waksman and 
Henrici, 1948 (Waksman, S. A. and Curtis, 
R. BE: Soil Sar Ts 23. 1916: S132 71919). 

Morphology: Long sporophores produce 
few imperfect spirals. Spores spherical, 0.75 
to 1.0 uw in diameter. 

Sucrose nitrate agar: 
smooth, gray, becoming brown with purplish 
tinge; center raised, margin yellow. Aerial 
mycelium dark brown to dark gray. 

Glucose-asparagine agar: Growth abun- 
dant, gray, becoming brown to dark brown. 

Nutrient agar: Growth gray to brownish, 
becoming dark brown, almost black. Soluble 


Growth restricted, 


pigment dark brown. Melanin-positive. 

Potato: Growth orange to orange-red. 

Gelatin: Surface growth slow, brownish. 
Liquefaction slow. 

Milk: Dark brown ring. Coagulation and 
slow peptonization. 

Starch media: Colonies small, dark brown. 
Slight hydrolysis. 

Cellulose: Moderate growth. 

Sucrose: Inversion. 

Nitrate reduction: Negative. 

Production of H.S: Negative. 

Temperature: Optimum 25°C. 

Antagonistic properties: Active against 
various bacteria. 

Habitat: Soil. 

Type culture: IMRU 3343. 

201. Streptomyces putrificus (Nikolaieva, 
1915) Waksman (Nikolaieva, E. Arch. Biol. 
Nauk. 18: 229, 1914). 


264 


Morphology: Sporophores spiral-shaped. 
Spores spherical. 

Nutrient agar: Growth colorless to gray- 
ish. Aerial mycelium white. No soluble pig- 
ment. 

Potato: Growth folded, sulfur-yellow. 
Aerial mycelium chalk-white. No soluble 
pigment. Melanin-negative. 

Milk: Pellicle heavy. Aerial mycelium 
white. Peptonization gradual without pre- 
vious coagulation. 

LoefHer’s serum: Growth yellow. No aerial 
mycelium. Serum liquefied and colored yel- 
lowish-brown. 

Odor: Strong, putrefactive. 

Habitat: Spring water. 

Remarks: Decomposes proteins ener- 
getically, with the formation of bad-smelling 
products (HS, NH;). Morphological prop- 
erties given by Krassilnikov (1949), who 
considers this organism as a member of the 
A. albus group. 


202. Streptomyces pyridomyceticus Okami 
et al., 1957 (Okami, Y., Maeda, K., and 
Umezawa, H. J. Antibiotics (Japan) 7A: 
55-56, 1954; 10A: 172, 1957). 

Morphology: Sporophores form flexible, 
open spirals. Spores of irregular size. 

Glycerol nitrate agar: Growth colorless 
to dark. Aerial mycelium thin, white, some- 
times gray to brownish-gray. No soluble 


pigment. 
Nutrient agar: Growth colorless. Aerial 


mycelium absent, or scant, white. No soluble 
pigment. 

Potato: Growth wrinkled, dark yellowish- 
brown. Aerial mycelium absent, or later 
white. No soluble pigment. 

Gelatin: Growth colorless. Aerial myce- 
lium white, sometimes grayish. No soluble 
pigment. No liquefaction. 

Starch: Hydrolysis. 

Milk: Growth yellowish, in the form. of 
surface ring. No aerial mycelium. Coagula- 
tion and peptonization absent or very slow. 

Blood: No hemolysis. 


THE ACTINOMYCETES, Vol. II 


Nitrate reduction: Negative. 

Carbon utilization: Utilizes 
dextrin, fructose, galactose, glucose, glycerol, 
maltose, xylose, and sucrose. Does not utilize 


arabinose, 


dulcitol, esculin, inulin, lactose, mannose, 
raffinose, rhamnose, salicin, sorbitol, sodium 
citrate, sodium acetate, and sodium suc- 
cinate. 

Habitat: Soil in Japan. 

Antagonistic properties: Produces anti- 
biotic pyridomycin. 

temarks: Isolated by means of chlortetra- 
cycline-containing agar. Related to S. cacaoz 
and S. flocculus, as well as to S. hygroscopicus. 
Above description was first given under the 
name S. albzdofuscus. It was later found that 
this name was preempted by Neukirch and 
Berestnew, and was, therefore, changed to 
S. pyridomyceticus. 


203. Streptomyces rameus Okami et al., 
1959) (Okami, Y., Suzuki, M., Takita, T., 
Ohi, IK., and Umezawa, H. J. Antibiotics 
(Japan) 12A: 257-262, 1959). 

Morphology: Aerial mycelium forms in- 
complete spirals or loops or hooks. Spores 
oval to oblong. 

Glycerol nitrate agar: Growth yellow. 
Aerial mycelium white. Soluble pigment 
absent or yellowish. 

Glucose-asparagine agar: Growth yellow- 
ish. Aerial mycelium white. Soluble pig- 
ment absent or yellowish. 

Calcium malate agar: Growth colorless to 
yellowish. Aerial mycelium off-white. No 
soluble pigment. 

Nutrient agar: Growth colorless to brown- 
ish. Aerial mycelium white. Soluble pigment 
absent or shght brownish. 

Starch agar: Growth colorless to yellowish. 
Aerial mycelium white. Soluble pigment 
yellowish. Hydrolysis weak to medium, 

Potato: Growth brownish. Aerial myce- 
lium white. Soluble pigment brown to black. 

Gelatin: Growth brownish. Aerial myce- 
lium white. Soluble pigment brown. Liquetac- 
tion doubtful. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Milk: Coagulation weak; peptonization 
doubtful. 

Nitrate reduction: Negative. 

Carbon sources: Utilizes arabinose, dex- 
trin, fructose, galactose, glucose, glycerol, 
inositol, maltose, mannitol, mannose, rafh- 
nose, starch, and sucrose. Lactose and xylose 
gave less response. Poor growth on inulin, 
rhamnose, salicin, sorbitol, sorbose, sodium 
acetate, and sodium citrate. 

Antagonistic properties: Produces strepto- 
mycin. 

ftemarks: Related to S. alboflavus, S. 
xanthophaeus, and S. orventalis. 


204. Streptomyces ramnait Bhuiyan and 
Ahmad, 1956 (Bhuiyan, A. M. and Ahmad, 
K. Ann. Biochem. Exptl. Med. India 16: 
101-104, 1955). 

Morphology: Open spirals, with 2 or 3 
turns. Spores spherical, 0.8 « in diameter. 
nitrate Growth whitish. 
Aerial mycelium white to pale rose. No 
soluble pigment. 

Glucose-asparagine agar: 


Sucrose 


agar: 


Growth color- 
less to pale rose. Aerial mycelium white, 
later pale rose. 

Calcium malate agar: Growth smooth, 
cream-colored. Aerial mycelium white. Me- 
dium becomes clear. 

Nutrient agar: Growth cream-colored, be- 
coming light brown. Aerial mycelium pow- 
dery, white. Soluble pigment shght brown 
coloration. Melanin-negative. 

Starch agar: Growth 
yellowish brown. No aerial mycelium. No 
soluble pigment. Hydrolysis rapid. 


cream-colored to 


Potato: Growth abundant, cream-colored. 
Aerial mycelium white, turning pale rose. 

Potato nutrient agar: Growth rapid, color- 
less to cream-colored. Aerial mycelium deep 
rose. Soluble pigment deep reddish-brown 
to almost red. 

Gelatin: Growth cream-colored. No aerial 
mycelium. Soluble pigment absent or light 
brown. Liquefaction medium. 


Milk: Growth cream-colored. No aerial 


mycelium. Coagulation, followed by 
tonization. Reaction acid. 
Nitrate reduction: Positive. 
Cellulose: Growth good. 


pep- 


Optimum temperature: 37°C. 
Antagonistic properties: Produces 
biotic ramnacin. 


anti- 


205. Streptomyces ramulosus Ettlinger et 
al., 1958 (Ettlinger, L., Giumann, E., Hiit- 
ter, R., IKeller-Schierlein, W., Kradolfer, 
F., Neipp, L., Prelog, V., and Zahner, H. 
Helv. Chim. Acta 41: 216-219, 1958). 

Morphology: Sporophores monopodially 
branched, straight with many side branches. 
Spores smooth (PI. IT h). 

Glycerol nitrate agar: Growth at first 
‘armine-red, later turning greenish-brown. 
Aerial mycelium ash-gray with greenish 
tinge. Substrate pigmented carmine-red. 

Glucose-asparagine agar: Growth yellow- 
ish-red. Aerial mycelium gray. Substrate 
carmine-red. 

Calcium malate agar: Growth hght vellow. 
Aerial mycelium chalky white to gray with 
greenish tinge. 

Glucose-peptone agar: Growth yellowish- 
red, partly greenish to greenish-black. Aerial 
mycelium powdery, Substrate 
greenish to brownish-black. 

Starch agar: Growth light yellow. Sub- 
strate ight carmine. Gradual hydrolysis. 

Potato: Growth yellowish-red. Aerial my- 
celium chalk-white to ash-gray. Substrate 
carmine-red. 

Gelatin: Growth light yellow. Soluble pig- 
ment light brown. No liquefaction. 

Milk: Light yellow pellicle. No aerial 
mycelium. Coagulation limited; peptoniza- 
tion good. 


ash-gray. 


Carbon. utilization: Glucose, L-xylose, p- 
inulin, well 
utilized. Does not utilize raffinose, L-arabi- 
p-mannitol, Question- 
able utilization of L-rhamnose, salicin. 


fructose, sucrose, p-sorbitol 


mesoinositol. 


nose, 


Antagonistic properties: Produces anti- 
biotic acetomycin, active against gram-posi- 


266 


tive and gram-negative bacteria, as well as 
against trichomonads and amoebae. 


206. Streptomyces resistomycificus Linden- 
bein, 1952 (Lindenbien, W. Arch. Mikro- 
biol. 17: 361-883, 1952). 

Morphology: Sporophores 
curling tips. Spores short, oval. 


long, with 

Glycerol nitrate agar: Growth yellow- 
brown to dark brown. Aerial mycelium ash- 
gray. Soluble pigment red-brown. 

Glucose-asparagine agar: Growth yellow- 
brown. Aerial mycelium ash-gray. Soluble 
pigment yellow-brown. 

Glycerol malate agar: Growth dark brown. 
Aerial mycelium ash-gray to red-gray. 
Soluble pigment gray to dark brown. 

Nutrient agar: Growth dark brown. 
Aerial mycelium absent or lead-gray. Soluble 
pigment dark brown. Melanin-positive. 

Glucose-peptone agar: Growth dark 
brown. Aerial mycelium white. Soluble pig- 
ment reddish to dark brown. 

Starch agar: Growth light yellow to red- 
dish-brown. Aerial mycelium gray-white, 
later red-gray. Soluble pigment lacking or 
reddish-brown. Hydrolysis strong. 

Potato: Growth brownish-black. Aerial 
mycelium reddish-white. Soluble pigment 
dark brown. 

Gelatin: Growth dark brown. Aerial my- 
celium white-gray. Soluble pigment chest- 
nut-brown. Good liquefaction. 

Milk: Growth dark brown. Aerial myce- 
lium white, later yellowish-red. Soluble pig- 
ment dark brown. Peptonization none or 
slight. 

Cellulose: No growth. 

Antagonistic properties: Produces resisto- 
mycin, which is active against gram-positive 
bacteria. 

Remarks: Gause et al. (1957) have de- 
scribed certain closely related forms, such as 
A. griseorubiginosus with a variety spiralis, 
and A. variabilis with a variety roseolus. 

Type culture: IMRU_ 3658. 


THE ACTINOMYCETES, Vol. II 


207. Streptomyces reticuli (Waksman and 
Curtis, 1916; Waksman, 1919) Waksman and 
Henrici, 1948 (Waksman, 8. A. and Curtis, 
R. E. Soil Sci. 1: 118, 1916; Waksman, 8. A. 
Soil Sci. 8: 143, 1919). 

Morphology: Aerial mycelium gives rise 
to simple verticils. Sporophores straight or 
spiral-shaped (sinistrorse) on different media. 
Spores spherical or oval, smooth, 1.0 to 1.4 
uw in diameter (PI. I a). 

Sucrose nitrate agar: Growth colorless, 
with yellowish tinge, becoming brownish. 
Aerial mycelium thin, cottony, white to ash- 
gray. No soluble pigment. 

Glycerol malate agar: Growth colorless. 
Aerial mycelium yellowish. No soluble pig- 
ment. 

Nutrient agar: Growth wrinkled, gray, 
becoming brownish. No aerial mycelium. 
Soluble pigment dark brown. 

Potato: Growth gray, with black center. 
Aerial mycelium ash-gray. Soluble pigment 
black. 

Gelatin: Growth gray to brown. Aerial 
mycelium white. Soluble pigment faint 
brown to dark brown. Good liquefaction. 

Milk: Coagulation rapid; peptonization 
slow. 

Starch: Growth brownish-gray. Hydrol- 
ysis. 

Cellulose: Scant growth. 

Nitrate reduction: Positive. 

Production of H.S: Positive. 

Invertase: Positive. 

Temperature: Optimum 25°C. 
properties: 
produce neomycin or a neomycin-like sub- 
stance. Some strains reduce double bonds in 


Antagonistic Some strains 


certain steroids. 

Habitat: Soil. 

Remarks: According to Ettlinger et al. 
(1958), the verticils are both primary and 
secondary; no spirals were observed. They 
also report the species to be melanin-nega- 
tive. One wonders whether they had a typi- 
cal culture. This culture was later found to 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


be identical with S. abzkoensum. Sakagami 
et al. (1958) described a variety latumcidicus 
that produced no aerial mycelium on most 


media and formed the antibiotic latumeidin. 
Type culture: IMRU 3344. 


208. Streptomyces rimosus Sobin et al., 
1950 (Sobin, B. A., Finlay, A. C., and Kane, 
J. H. U.S. 2,516,080, July 18, 1950; see also 
Kochi, M., eé al. Proc. Natl. Acad. Sci. U.S. 
38: 383-391, 1952). 

Morphology: Sporophores long, usually 
straight, occasionally open or closed spirals 
depending on composition of medium. Speres 
cylindrical, 0.6 to 0.7 by 0.8 to 1.4 ». A 
microscopic study of S. rzmosus (strain 3558) 
grown on yeast extract-glucose agar, after 
14 days incubation, revealed the following: 
Aerial hyphae were long and fairly straight, 
segmenting into chains of even, bead-like 
spores. Other aerial hyphae were 
tangled, branching, twisting into spirals, 
also segmented into chains of bead-like 


long, 


spores. 

Sucrose nitrate agar: Growth thin, cream- 
colored, developing slowly at first, later be- 
coming abundant, much folded or lichenoid; 
reddish-brown to orange. Aerial mycelium 
appears first over the drier edge of the 
growth or in the form of thin white patches. 
When the culture becomes older, a faint 
bluish zone appears around the edge of the 
growth. Soluble pigment faint yellowish. 

Glucose-asparagine agar: Growth at first 
cream-colored, becoming brownish to orange- 
brown with age. Aerial mycelium white. 
Soluble pigment yellowish to golden. 

Yeast-glucose agar: Growth much more 
rapid than in synthetic media; lichenoid, 
cream to brownish. Aerial mycelium appears 
at an early stage of growth, white, later 
tending to become mouse-gray. Soluble pig- 
ment yellowish. 

Nutrient agar: Growth poor, cream- 
colored to yellowish-brown to mouse-gray. 
Aerial mycelium white or absent. Soluble 
pigment absent or yellowish. 


267 
Starch Growth limited, cream- 
colored, with deeper brown center. No aerial 
mycelium. Limited hydrolysis. 


agar: 


Potato: Growth lichenoid, cream-colored 
to reddish-brown. Aerial mycelium white to 
gray to dark brown. Soluble pigment yel- 
lowish-brown. 

Gelatin: Growth cream-colored to brown- 
ish. Aerial mycelium white. Slow liquefac- 
tion. No soluble pigment, only a faint yel- 
lowish coloration of liquefied portion. 

Milk: Heavy pellicle, 
colored to yellowish. Aerial mycelium gray- 
ish-white. Peptonization, without coagula- 
tion. 


surface cream- 


Cellulose: No growth. 

Nitrate reduction: Positive. 

Production of HS: Negative. 

Antagonistic properties: Produces an anti- 
bacterial antibiotic, oxytetracycline, and an 
antifungal agent, rimocidin. 

Habitat: Soil. 

temarks: A variety of S. rimosus (forma 
paromomycinus) Was briefly described (Brit. 
Pat. 797,568, July 2, 1958). This variety was 
isolated from a soil in South America. It dif- 
fers from S. r¢mosus in certain minor cultural 
properties (somewhat lighter color on agar 
media) and in poorer utilization of arabinose. 
Both form dense clusters of spirals on various 
synthetic media and on glucose-tryptone 
agar. The variety produces an antibiotic, 
paromomycin, apparently closely related to 
the neomycin group. 

Type culture: IMRU 3558; ATCC 10,970, 


209. Streptomyces rochei Berger et al., 
1949 (Berger, J., Jampolsky, L. M., and 
Goldberg, M. W. Arch. Biochem. 22: 476— 
478, 1949; Waksman, S. A. and Lechevalier, 
H. Guide to the classification and identifica- 
tion of the actinomycetes and their anti- 
bioties. The Wilhams and Wilkins Co., 
Baltimore, 1953, p. 40). 

Morphology: Sporophores straight, 1.5 yu 
in diameter; often, but not always spirally 
twisted; spirals usually short and loose with 


268 


rarely more than 2 to 3 coils. Spores oval to 
elliptical, sometimes spherical, 1.2 to 2.8 by 
OSxto. aa. 

Sucrose nitrate agar: Growth thin, color- 
less, covered with sandy lavender to dark 
gray aerial mycelium. Reverse of growth 
light gray, later becoming grayish-yellow. 
No soluble pigment. 

Nutrient agar: Growth cream-colored. 
Aerial mycelium white. No soluble pigment. 

Calcium malate glycerol: Growth good, 
raised in center. Aerial mycelium gray, buff 
around the edges, having a fuzzy appearance. 

Glucose agar: Growth smooth, yellowish, 
covered with white to gray aerial mycelium. 
Yellowish soluble pigment. 

Potato: Growth abundant, — lichenoid, 
cream-colored. Aerial mycelium abundant, 
cottony, white to gray. Color of plug be- 
comes reddish-tan. 

Gelatin: Cream-colored ring, covered with 
white aerial mycelum. Rapid liquefaction. 
Faint vellow soluble pigment. 

Milk: Cream-colored to brownish ring. 
Coagulation and rapid peptonization. 

Starch: Growth brownish. Aerial myce- 
lium mouse-gray. Diastatic action strong. 

Production of HS: Negative. 

Antagonistic properties: On certain com- 
plex nitrogenous media, the organism shows 
a wide range of antimicrobial activity, 
partly because of borrelidin. 

temarks: Morphologically, the culture 
resembles S. albidoflavus, S. californicus, S. 
lipmaniz, and certain others, but it is not 
believed to be identical to any of them. 
Ettlinger et al. (1958) considered this or- 
ganism as a strain of S. fradiae. Okami and 
Suzuki (1958) could not demonstrate any 
spirals on several media tested. 

Type culture: IMRU 3602; ATCC 10,739. 


210. Streptomyces roseochromogenes (Jen- 
sen, 1931) Waksman 1948 
(Jensen, H. L. Proe. N.S. 
Wales 56: 359, 1931). 

Waksman and Curtis (1916) and Waks- 


and Henrici, 


Linnean Soc. 


THE ACTINOMYCETES, Vol. II 


man (1919) described an organism as A. 
roseus IxXrainsky. This culture was, in con- 
trast to Krainsky’s organism, chromogenic. 
Jensen (1931) compared it with his own 
isolates and changed the name A. roseus to 
roseochromogenes, because of the fact that 
the name roseus had previously been used by 
Namyslowsky (1912). 

Morphology: Sporophores form numerous 
open and closed sinistrorse spirals; some- 
times 3 to 5 branches issue together from 
end point of main stem, giving impression 
of brooms or verticils. Spores spherical, 1.0 
to 1.2 by 1.3 to'3.0-¢ (PL. V, 2b). 

Sucrose nitrate agar: Growth thin, spread- 
ing, colorless to pale yellow. Aerial mycelium 
pale grayish-rose. 

Glucose-asparagine agar: pale 
yellow. Aerial mycelium white, later be- 
coming rose-cinnamon, with many small 
white tufts. 

Nutrient agar: Growth wrinkled, yellow- 
ish-gray, later brown-red. Aerial mycelium 
white, then rose-gray. Soluble pigment deep 


Growth 


brown. 

Potato: Growth wrinkled, yellowish-gray 
to grayish-black. Aerial mycelium absent or 
Soluble pigment black. Melanin- 
positive. 

Gelatin: Colonies small, cream-colored, in 
bottom of liquefied zone. Soluble pigment 
brown. Liquefaction medium. 

Milk: Coagulation limited; peptonization 


white. 


slow. 

Starch media: Growth colorless, spreading. 
Hydrolysis good. 

Nitrate reduction: Positive. 

Production of HS: Positive. 

Antagonistic properties: Active against 
various bacteria; produces antibiotic roseo- 


mycin. 
Habitat: Soil. 
Remarks: Jensen (1931) obtained, on 


plating the tufts of white aerial mycelium 
arising on agar media, a variant with pure 
white aerial mycelium. 

Type culture: ATCC 13,400. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


211. Streptomyces roseocitreus Kato, 1953 


(Kato, H. J. Antibiotics (Japan) 6A: 143; 
6B: 206-208, 1953). 
Morphology: Sporophores produce nu- 


merous open and closed spirals of the dex- 
trorse type. Spores oval, 1.2 to 1.5 by 1.6 to 
1.8 p. 

Sucrose nitrate agar: Growth pale olive- 
buff, later changing to deep olive-buff, ivory- 
yellow, or colonial buff. Aerial mycelium 
scant, white. Soluble pigment at first faint 
creamy, later changing to colonial buff. 

Glycerol-calctum malate agar: Growth at 
first transparent with gray to blackish-blue 
patches, later becoming light yellowish- 
olive to reed-yellow. Aerial mycelium thin, 
white, at first having tinge of gray. Soluble 
pigment yellowish with tinge of green. 

Nutrient agar: Growth olive-buff, later 
changing to deep olive-buff with bluish 
patches. No aerial mycelium. Soluble pig- 
ment brown. 

Starch agar: Growth hyaline, cottony, 
reverse becoming faint bluish. Aerial myce- 
lium white, later becoming livid pink, and 
finally pale grayish-vinaceous. Enzymatic 
zone fair to good. 

Potato: Growth thick, folded, pale olive- 
buff, later deep olive to dark olive. Aerial 
mycelium at first white, later becoming livid 
pink to vinaceous-buff. Color of plug black- 
ish-brown. 

Gelatin: Whitish colonies on surface of 
tube. Aerial mycelium scant, white. Soluble 
pigment brown. Liquefaction slow. 

Milk: Growth in yellow ring with patches. 
Soluble pigment yellowish. No coagulation; 
peptonization slow. 

Cellulose: No growth. 

Carbon utilization: Utilizes 
arabinose, b-sorbitol, salicin, and sodium 


sucrose, L- 


acetate; not sodium succinate. 

Antagonistic properties: Produces anti- 
biotics roseocitrin A and B. 

Habitat: Soil. 


212. Streptomyces roseodiastaticus (Duché, 


269 


1934) nov. comb. (Duché, J. Les actinomyces 
du groupe albus. P. Lechevalier, Paris, 
p. 329, 1934). 

Morphology: Growth consists of fine my- 
celium 0.5 to 0.7 uw in diameter. Aerial my- 
celium of larger diameter, but usually less 
than 1 py. 


Glucose nitrate agar: Growth limited, 
cream-colored, becoming white with a 


brownish reverse; on prolonged incubation 
the culture becomes rose-gray. Soluble pig- 
ment brownish. 

Glycerol nitrate agar: Growth cream- 
colored, becoming rose-violet; reverse red. 

Asparagine agar: Growth cream-colored, 
becoming rose-white; reverse of growth 
reddish-brown. Soluble pigment vellow. 

Tyrosine medium: Pigment 
later becoming brown. 


brownish, 


Gelatin: Liquefied. No soluble pigment. 
Melanin-negative. 

Potato: Growth cream-colored, becoming 
brownish-white. Soluble pigment 
only in presence of glycerol. 

Milk: Growth limited. 
slow. 

Starch: Diastatic action weak. 

temarks: Closely related to S. halstedii 
and considered as a transitional form. Ac- 


brown, 


Peptonization 


cording to Ettlinger et al. (1958), this or- 
ganism is related to S. griseus. 


213. Streptomyces roseoflavus Arai, 1951 
(Arai, T. J. Antibiotics (Japan) 4: 215-221, 
1951). 

Morphology: Sporophores form spirals. 
Spores oval to oblong, 0.8 to 1.0 by 1.0 to 
LS. 

Sucrose nitrate agar: Growth colorless to 
yellowish. Aerial mycelium powdery, white 
to yellow-rose. 

Glucose-asparagine agar: Growth color- 
less to yellowish-white. Aerial mycelium 
rose-colored. 

Nutrient Growth folded, 
white-gray to golden yellow. Aerial myce- 


agar: much 


Ficure 44. Verticil formation by S. roseover- 
ticillatus (Reproduced from: Shinobu, R. Mem. 
Osaka Univ. Ser. B, No. 5, p. 938, 1956). 


lium limited to center of colonies, white to 
rose. 

Starch agar: Growth golden yellow. Aerial 
mycelium whitish. 

Potato: Growth yellow. No aerial myce- 
lium. No soluble pigment. 

Gelatin: Liquefaction strong. Colonies at 
bottom of liquefied zone orange-brown. No 
soluble pigment. Melanin-negative. 

Milk: Ring cream-colored. Coagulation 
and peptonization rapid, medium becoming 
strongly alkaline. 

Cellulose: Growth on paper fair; cellulose 
decomposed. 

Nitrate reduction: Positive. 

Production of HS: Negative. 

Antagonistic properties: Produces a basic 
antibiotic, flavomycin, similar to neomycin. 

temarks: Culture similar to S. micro- 
flavus. Gause et al. (1957) described other 
closely related cultures, such as A. roseoful- 
vis. 

Type culture: IMRU 3672; ATCC 13,167. 

214. Streptomyces roseoverticillatus Shin- 
obu, 1956 (Shinobu, R. Mem. Osaka Univ., 
B (N.S8.) 5: 84-93, 1956). 

Morphology: Sporophores produce abun- 


THE ACTINOMYCETES, Vol. II 


dant primary and secondary verticils (Fig. 
44). Spores spherical to elliptical, 0.8 to 1.0 u. 

Sucrose nitrate agar: Growth thin, mod- 
erate, pinkish-red. Aerial mycelium cottony, 
pink to pale pink. 

Glucose-asparagine agar: Growth red to 
purple-red. Aerial mycelium cottony, dull 
red to reddish-brown. 

Nutrient agar: Growth reddish-brown to 
deep brown. Aerial mycelium thin, pinkish 
to red. Soluble pigment reddish-brown to 
deep brown. 

Potato: Growth brownish-red to dull red. 
Aerial mycelium powdery, pink to reddish- 
purple. Soluble pigment brown. 

Milk: Growth red. Aerial mycelium pale 
pink. Coagulation and peptonization strong. 
Soluble pigment pale brown. 

Gelatin: Rapid liquefaction. 

Starch: Rapid hydrolysis. 

Tyrosinase reaction: Weak. 

Cellulose: No growth. 

Nitrate reduction: Positive. 

Carbon utilization: Utilizes fructose; 
inositol uncertain. Does not utilize xylose, 


rhamnose, sucrose, lactose, raffinose, or 
mannitol. 


Habitat: Soil in Japan. 

Remarks: Resembles S. rubrireticult. 

215. Streptomyces roseus (Namyslowsky, 
1909; emend. Krainsky, 1914; emend. Waks- 


man and Curtis, 1916) Waksman and 
Henrici, 1948 (Namyslowsky, B. Centr. 


Bakteriol. Parasitenk. Abt. I, Orig. 62: 564, 
1909; Krainsky, A. Centr. Bakteriol. Para- 
sitenk. Abt. II, 41: 682-683, 1914; Waks- 
man, 8. A. and Curtis, R. E. Soil Sci. 1: 125, 
1916). 

Morphology: Sporophores produce num- 
erous open and closed dextrorse spirals. Ac- 
cording to Okami, sporophores are straight, 
without spirals. Spores oval to elongate, 1.5 
to 2.0 by 1.1 wu. 

Sucrose nitrate agar: Growth colorless. 
Aerial mycelium pale brownish-vinaceous. 
No soluble pigment. 


DESCRIPTION OF SPECIES OF STREPTOMYCES Prt 


Glycerol malate agar: Growth colorless. 
Aerial mycelium white to rose. 

Nutrient agar: Growth white, turning 
yellowish. No aerial mycelium. No soluble 
pigment. 

Starch agar: Growth colorless. Aerial my- 
celium white with shade of pink. No soluble 
pigment. Hydrolysis medium. 

Gelatin: Growth yellowish-brown. Aerial 
mycelium white. Soluble pigment brown. 
Liquefaction slow; in some cases no liquefac- 
tion. Melanin-negative. 

Potato: Growth brownish. No aerial my- 
celium. Soluble pigment brownish or absent. 

Milk: No coagulation; gradual peptoniza- 
tion. 

Cellulose: No growth. 

Invertase: None. 

Nitrate reduction: Rapid. 

Habitat: Soil. 

Remarks: Various cultures have 
described under this name. Krassilmkov 
(1949) considered it as a varietal strain of 
S. ruber. 

Type culture: IMRU 3772. 


been 


216. Streptomyces ruber (KKrainsky, 1914) 
Waksman and Henrici, 1948 (Ikrainsky, A. 
Centr. Bakteriol. Parasitenk. Abt. II., 41: 
649-688, 1914). 

Not Actinomyces ruber Ruiz-Cazabo, 1894; 
not Actinomyces ruber (<xruse, 1896) San- 
felice, 1904. 

Morphology: Sporophores straight, 
branching; a few spirals may be formed. 
Spores spherical and oval, 0.7 to 0.8 by 0.8 
to 1.0 p. 

Sucrose nitrate agar: Growth abundant, 
orange to coral-red. Aerial mycelium red to 
red-orange to dark red. Pigment insoluble 
unless vegetable oil present in medium. 

Nutrient agar: Growth elevated, wrinkled, 
olive-green. No aerial mycelium. 

Glucose agar: Growth abundant, coral-red. 

Potato: No growth. 

Gelatin: Growth yellow, flaky. 
Liquefaction slow, increasing with growth. 
Melanin-negative. 


scant, 


Milk: Dark ring with red tinge. Coagula- 
tion; peptonization gradual. 

Starch: Hydrolysis weak. 

Sucrose: Inversion positive. 

Cellulose: Growth in form of red spots. 

Nitrate reduction: Positive, depending on 
carbon source. 

Production of H.S: Negative. 

Temperature: Optimum 37°C. 

Pigments: Soluble in organic solvents; 
alcohol extracts a red-violet pigment and 
petroleum-ether a red-orange pigment (Kriss, 
1956). 

Antagonistic properties: Strongly effective 
upon gram-positive bacteria. Various anti- 
biotics are produced by different strains. 

Habitat: Soil. 

Remarks: Above description is based 
largely upon that given by Krainsky. Closely 
related forms include A. longisporus ruber 
Krassilnikov, which is said to form some- 
what longer spores, and to give sometimes a 
brown coloration in protein media. A. auran- 
trogriseus Gause et al. also appears to be 
closely related. 

217. Streptomyces rubescens (Jarach, 1931) 
Umezawa et al., 1952 (Jarach. Boll. sez. ital. 
soc. intern. microbiol. 3:48, 1931; Umezawa, 
H., Tazaki, T., and Fukuyama, 8. J. Anti- 
biotics (Japan) 5: 469, 1952). 

Morphology: Sporophores short, curved, 
well branched; no spirals. Spores spherical or 
oval. 
first 
white, changing to salmon-pink. Aerial my- 


Sucrose nitrate agar: Growth at 
celium powdery, white. No soluble pigment. 

Nutrient agar: Growth same as on sodium 
nitrate agar. 


Blood 


growth becomes salmon-pink. Aerial myce- 


agar: After 10 days’ incubation, 


lium powdery, white. No soluble pigment. 
No hemolysis. 
Egg media: Growth colorless, changing to 
coral-pink. Aerial mycelium powdery, white. 
Potato: Growth coral-pink. Aerial myce- 
lium powdery, white. Plug changes shghtly 


272 


to brown. No soluble pigment. Melanin- 
negative. 

Gelatin: Surface growth coral-pink. No 
liquefaction. No soluble pigment. 

Milk: Growth coral-pink. Aerial mycelium 
powdery, white. No coagulation and no 
peptonization. Soluble pigment sometimes 
slightly reddish. 

Starch: No hydrolysis. 

Nitrate reduction: None. 

Production of HS: Negative. 

Carbon utilization: Glycerol and glucose 
utilized, but not other carbohydrates. 

Antagonistic properties: Produces an anti- 
viral agent, abikoviromycin. 

Remarks: A. griseoruber of Gause et al. 
(1957) appears to be a closely related form. 
This organism is considered by R. Gordon as 
a Nocardia, related to N. asteroides. 

Type culture: IMRU 3655. 


218. Streptomyces rubrireticuli (Waksman, 
1919) Waksman and Henrici, 1948 (Waks- 
man, 8. A. Soil Sci. 8: 146, 1919). 

Synonyms: Actinomyces 
Waksman, 1919; A. 
ed., 1925. 

Morphology: Sporophores produce both 
primary and secondary verticils; composi- 
tion of medium influences structure of 
sporophores, glucose-asparagine agar favor- 
ing spiral formation. Spores oval-shaped, 
smooth (PI. I a). 

Sucrose nitrate agar: Growth abundant, 
spreading, usually pink. Aerial mycelium 


reticulus-ruber 
reticulus Bergey, 2nd 


white, later rose to pink. 

Glucose-asparagine agar: Entire growth 
abundant, spreading, rose-red. 

Nutrient agar: Growth red, with yellowish 
margin, becoming red. Soluble pigment dark 
brown. 

Starch agar: Growth white with red tinge. 
Hydrolysis fair. 

Potato: Growth cream-colored, later pink 
to dark red. Melanin-positive. 

Gelatin: Surface growth yellowish-red to 
pink. Ready liquefaction. Brown pigment. 


THE ACTINOMYCETES, Vol. II 


Milk: Growth abundant. Coagulation and 
peptonization. 

Invertase: Positive. 

Cellulose: Growth good. 

Nitrate reduction: Rapid. 

Production of H.S: Positive. 

Antagonistic properties: Certain strains 
produce an antibiotic designated as streptin; 
others produce trichonin. 

Habitat: Soil. 

Remarks: Numerous cultures that pro- 
duce a rose to pink substrate growth, a 
soluble brown pigment in organic media, and 
both primary and secondary verticils in the 
sporophores have been described. It is suf- 
ficient to mention A. biverticillatus by Gause 
et al. (1957). 

Type culture: IMRU 3631. 


219. Streptomyces rutgersensis (Waksman 
and Curtis, 1916) Waksman and Henrici, 
1948 (Waksman, 8S. A. and Curtis, R. E. 
Soil Sei. 1: 123, 1916; 8: 152, 1919). 

Morphology: Sporophores produce abun- 
dant close and open spirals. Spores spherical 
and oval, 1.0 to 1.2 uw, with tendency to bi- 
polar staming. 

Sucrose nitrate agar: Growth thin, color- 
less, becoming brownish to almost black. 
Aerial mycelium white, becoming dull gray. 

Glucose-asparagine agar: Growth abun- 
dant, brown, becoming black with cream- 
colored margin. No aerial mycelium appears 
within 15 days. 

Nutrient agar: Growth thin, wrinkled, 
cream-colored. 

Starch agar: Growth gray, spreading. 
Hydrolysis good. 

Potato: Growth abundant, much folded. 
Aerial mycelium white-gray. Melanin-nega- 
tive. 

Gelatin: Growth cream-colored. Liquefac- 
tion medium. No soluble pigment. 

Milk: Cream-colored Coagulation 
and slow peptonization. 

Cellulose: Growth seant. 


ring. 


Sucrose: Inversion weak. 


DESCRIPTION OF SPECIES OF STREPTOMYCES Pa les 


Nitrate reduction: Good. 

Production of H.S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Various strains 
produce xanthomycin-like substances; others 
produce ruticin. 

Remarks: The pigment formed is not 
soluble. Krassilmikov (1949) considered the 
organism, quite incorrectly, as a variety of 
A. chromogenes. 

Type culture: IMIRU 3350. 

220. Streptomyces sahachirot Hata et al., 
1954 (Hata, T., Koga, F., Sano, Y., Kana- 
mori, K., Matsumae, A., Sugawara, R., 
Hoshi, T., and Shima, T. J. Antibiotics 
(Japan) 7A: 107-112, 1954). 

Morphology: Sporophores form numerous 
closed spirals with a few open spirals. Spores 
oval or cylindrical, 0.7 to 1.8 by 0.5 to 0.8 uw. 

Sucrose nitrate agar: Growth folded, dark 
orange. Aerial mycelium velvety, white to 
pale grayish-brown. Soluble pigment  yel- 
lowish-brown. 

Calcium malate agar: Growth thin, cream- 
colored with orange-yellow reverse. Aerial 
mycelium thin, white, powdery. Soluble pig- 
ment pale orange-yellow. 

Nutrient agar: Growth glistening, white- 
gray. No aerial mycelium. Soluble pigment 
light brown to yellow. 

Starch agar: Growth thin, yellowish-white. 
Aerial mycelium thin, powdery, pale red- 
brown. No soluble pigment. Slow hydrolysis. 

Potato: Growth wrinkled, pale yellowish- 
brown. Aerial mycelium thin, white. Soluble 
pigment absent or faint brown. 

Gelatin: Growth limited, white. No aerial 
mycelium. No soluble pigment. 

Milk: Surface growth white to pale yellow. 
Coagulation; no  peptonization. 
alkaline. Soluble pink pigment. 

Nitrate reduction: Positive. 

Carbon utilization: Xylose, 
lactose, trehalose, mannitol, sucrose, salicin, 
glucose, maltose, mannose, glycerol, dextrin, 
fructose, starch, galactose, sorbitol utilized. 


Strongly 


arabinose, 


Rhamnose, raffinose, inositol, esculin, dulci- 
tol, inulin, sodium acetate, sodium citrate. 
sodium succinate not utilized. 

Antagonistic properties: Produces anti- 
tumor agent carzinophilin. 

221. Streptomyces sampsonii (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Aerial mycelium produces 
long, straight sporophores, rarely spiral- 
shaped. Spores cylindrical, 0.8 to 1.0 by 0.5 
m (spores oval to spherical, Waksman and 
Gordon). 

Sucrose nitrate agar: Growth wrinkled, 
pale gray to white. Aerial mycelium very 
scant, white. Soluble pigment green to buff. 

Potato: Growth wrinkled, grayish. Aerial 
mycelium white. Soluble pigment golden 
brown (none, Waksman and Gordon). 

Gelatin: Surface growth scant, gray. 
Aerial mycelium trace, whitish. Liquefaction 
rapid. Melanin-negative. 

Milk: Surface growth good, whitish. No 
aerial mycelium. No coagulation; no pep- 
tonization (rapid peptonization, Waksman 
and Gordon). 

Starch: No hydrolysis (rapid hydrolysis, 
Waksman and Gordon). 

Nitrate reduction: Positive. 

Tyrosinase reaction: Negative. 

Temperature: 28°C, 

Habitat: Potato scab. 

Type culture: IMRU 3371. 


222. Streptomyces sayamaensis Arishima 
et al., 1955 (Arishima, M., Sekizawa, Y., Sato, 
T., and Miwa, K. 
29: 810-817, 1955). 
Morphology: Sporophores straight, spores 


short rods to cylindrical, 1.0 by 1.5 w. 


J. Agr. Chem. Soc. Japan 


Sucrose nitrate agar: Growth pale yellow. 
Aerial mycelium gray with brownish tinge. 
Soluble pigment yellow. 

Glucose-asparagine agar: Growth white to 
pale yellow, turning pale brown. Aerial my- 
celium white, becoming pale brown. 

Calcium malate Growth limited. 


agar: 


274 


Aerial mycelium gray. Soluble pigment 
brownish. 
Starch agar: Growth brownish-yellow. 


Aerial mycelium white, turning gray. Hy- 
drolysis. 

Nutrient agar: Growth pale orange-yellow. 
No aerial mycelium. 

Potato: Growth heavy, pinkish-gray with 
purplish tinge. Soluble pigment reddish- 
brown. 

Gelatin: Surface pellicle pale yellow. No 
soluble pigment. No liquefaction in 15 days 
at 26°C. Melanin-negative. 

Milk: Yellow-gray ring. Coagulation and 
peptonization. 

Nitrate reduction: Negative. 

Cellulose: No growth. 

Optimum temperature: 35-87°C. 

Tyrosinase reaction: Negative. 

Carbon utilization: Utilizes b-galactose, 
sucrose, maltose, sodium citrate and succi- 
nate; does not utilize xylose, arabinose, 
lactose, rhamnose, raffinose, inulin, manni- 
tol, sorbitol, inositol, and salicin. 

Antagonistic properties: Produces chlor- 
tetracycline. 

Habitat: Soil in Japan. 

temarks: Related to S. aureofaciens. 


THE ACTINOMYCETES, Vol. II 


999 


240. 


Streptomyces scabies (Thaxter, 1891) 
Waksman and Henrici, 1948 (Thaxter, R. 
Ann. Rept. Conn. Agr. Expt. Sta. 1891, p. 
153): 

Morphology: Sporophores much branched, 
wavy or slightly curved; occasionally form 
spirals. Spores cylindrical, 0.8 to 1.0 by 1.2 
to 1.5 w (Fig. 45). 

Sucrose nitrate agar: Growth abundant, 
wrinkled, raised, cream-colored. 
Aerial mycelium cottony, white to gray. 
Growth — re- 
stricted, folded, cream-colored. Aerial my- 


gray to 
agar: 


Glucose - asparagine 


celium scant, white to gray. 
Nutrient agar: Growth wrinkled, white to 


straw-colored, opalescent to opaque. No 
aerial mycelium. Soluble pigment deep 


golden brown. 

Potato: Growth gray, opalescent, becom- 
ing wrinkled, black. Aerial mycelium seant, 
erayish-white. Color of plug brown. 

Gelatin: Surface growth cream-colored, 
becoming brown. Liquefaction slow. Soluble 
pigment yellowish. 

Milk: Surface ring brown, with greenish 


tinge. Coagulation and peptonization lim- 
ited. 
Starch: Hydrolysis. 


Figure 45. Sporophores of S. scabies, showing 


of EK. Baldacci, University of Milan, Italy). 


chains of transparent spores, X 15,000 (Courtesy 


DESCRIPTION OF SPECIES OF STREPTOMYCES 215 


Sucrose: Inversion. 

Nitrate reduction: Positive. 

Tyrosinase reaction: Strong. 

Antagonistic properties: Certain strains 
show positive antimicrobial action; others 
are negative. 

Habitat: Numerous strains of this organ- 
ism have been isolated from various forms of 
potato scab and sugar beet scab throughout 
the world. True causative agent of scab. 

Remarks: According to Hoffmann (1958), 
growth on synthetic agar is reddish with 
dark gray aerial mycelium; on glucose agar, 
growth is colorless with blue-gray aerial my- 
celium; on asparagine agar, growth is dark 
red with no aerial mycelium. 

Closely related forms include S. clavzfer, 
S. spiralis, S. carnosus, and S. sampsonzi 
described by Millard and Burr; also A. 
xanthostromus and A. ochroleucus of Wollen- 
weber. Various strains differ in the amount 
of aerial mycelium produced and in their 
biochemical properties. 

Type culture: IMRU 3018. 


224. Streptomyces setonii (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight, wavy, 
formed in clumps. Spores oval, 0.6 to 0.8 by 
0.85 yu. 

Sucrose nitrate agar: Growth abundant, 
smooth, yellow to brown. Aerial mycelium 
gray to olive-buff. Soluble pigment faint 
yellowish to brown. 

Nutrient agar: Growth colorless. Aerial 
mycelium smooth, white. Soluble pigment 
brownish. Melanin-negative. 

Glucose agar: Growth lichenoid, gray to 
brown. Aerial mycelium abundant, white to 
olive-buff. Soluble pigment golden brown. 

Potato: Growth heavy, wrinkled, brown 
to red-violet. Aerial mycelium abundant, 
white to green to olive-buff. 

Gelatin: Surface growth gray. Aerial my- 
celium white. Rapid liquefaction. Soluble 
pigment brownish. 


Milk: Surface growth, covered with ring 
of white aerial mycelium. Questionable co- 
agulation, followed by rapid peptonization. 

Starch cream-colored. 
Aerial mycelium patchy, white. Hydrolysis. 

Cellulose: Growth colorless. 

Nitrate reduction: Positive. 

Temperature: Grows well at 37.5°C. 

Habitat: Scabby potatoes. 

Remarks: Millard and Burr also described 
a similar form under the name A. 


agar: Growth 


setonai 


flavus. Ettlinger et al. (1958) consider this 


organism as a strain of S. griseus. Hoffmann 
(1958) described an organism with light gray 
aerial mycelium as a strain of S. setoniz. 
Type culture: IMRU 3375. 


225. somaliensis (Brumpt, 
1906; emend. Erikson, 1935) Waksman 
(Brumpt, E. Arch. Parasitol. Paris 10: 489, 
1906; Precis de Parasitologie. Paris, 2nd ed., 
p. 967, 1913; Erikson, D. Med. 
Council (Brit.) Spee. 
1935, p:. 17-18). 

Morphology: Substrate growth made up of 
simple branching, unicellular mycelium with 
long straight filaments. Aerial mycelium 


Streptomyces 


Xesearch 
Rept. Ser. No. 203: 


forms straight nonsegmented sporophores 
with typical chains of spores, 
diameter. 


20 M in 
Glucose-asparagine agar: Growth thin, 
smooth, and soft. 

Glycerol nitrate agar: Growth abundant, 
colorless to dark gray and black. 

Nutrient agar: Growth abundant, granu- 
lar, yellowish, with small discrete colonies at 


margin; later growth colorless, colonies 
umbilicated. 


Potato: Colonies round and oval, partly 
piled up in rosettes. Aerial mycelium whitish- 
gray. Plug discolored. Later, aerial myce- 
hum becomes transient, growth nearly black. 

Blood agar: Growth in form of small, dark 
found and umbilicated, 


brown. colonies. 


piled up in confluent bands. Reverse red- 
black. Blood hemolyzed. 


Dorset’s egg medium: Growth colorless, 


276 


becoming opaque, cream-colored, very wrin- 
kled. Later, rough, yellow; medium lique- 
fied. 
Gelatin: Black 
sediment at bottom. Rapid liquefaction. 
Milk: Surface pellicle heavy, wrinkled. 
Milk coagulated and completely peptonized. 
Starch: Hydrolysis. 


Growth cream-colored. 


Habitat: Frequently found in Africa. 
femarks: Although S. somaliensis has 


long been known, there has been, until re- 
cently, no detailed description of the or- 
ganism beyond the fact that it possesses 
around the grain a distinctly hard sheath 
which is insoluble in potash and eau de 
javelle. The rare occurrence of septa and 
occasional intercalary chlamydospores 1s re- 
ported by Brumpt, but has not been con- 
firmed by Erikson. Chalmers and = Chris- 
topherson merely mentioned the growth on 
potato as yellowish-white and lichenoid, 
without describing any aerial mycelium. 
According to Mariat, S. somaliensis hy- 
drolyzes gelatin, serum albumin, and egg 
albumin; utilizes casein hydrolyzate, but not 
urea, (NH,).SO, and KNO; as 
sources; utilizes glucose, maltose, and fruc- 


nitrogen 


tose, but not xylose, starch, mannitol, or 
paraffin as carbon sources. 


226. Streptomyces spectabilis Dietz, 1957* 
(Brit. Pat. 811,757, April 8, 1959; Am. Rev. 
Tuberc. 75: 576, 1957). 

Morphology: Sporophores monopodially 
branched, long, straight. Pigment granules 
produced in both substrate and aerial myce- 
hum. 

Sucrose 


nitrate Growth mottled 


orange to cream-orange. Aerial mycelium 


agar: 


mottled orange to orange. 
Starch-nitrate agar: Growth cream-colored, 
flecked with orange. Aerial mycelium pale 
pink to orange. Starch hydrolyzed. 
Starch-peptone-beef extract agar: Growth 
cream-colored, turning orange. Aerial myce- 


* Personal communication. 


THE ACTINOMYCETES, Vol. II 


lium deep orange to pale pink. Soluble pig- 
ment yellow. 

Gelatin: Medium liquefaction. Soluble 
pigment slightly yellow to dark brown. 
Melanin-negative. 

Milk: Growth orange. Soluble pigment 
brown. Peptonization varies with strain. 
Acid formation by some strains. 

Carbon utilization: Utilizes various sugars 
and organic acids, depending on strain. Does 
not utilize rhamnose, sucrose, inulin, ducitol, 
p-sorbitol, fumarates, oxalates, or salicylates. 

Nitrate reduction: Negative. 

Production of H.S: Positive. Some strains 
negative. 

Antagonistic properties: Produces antibi- 
otic streptovaricin. 

Remarks: Closely related to S. fulvissemus. 


227. Streptomyces spheroides Wallick et al., 
1955 (Wallick, H., Harris, D. A., Reagan, 
M. A., Ruger, M., and Woodruff, H. B. 
Antibiotics Ann. 1955-1956, p. 909-917). 

Morphology: Sporophores form spirals, 
the majority of which are closed and com- 
pact; in some areas the spirals appear ball- 
like. Spores oval, 0.7 to 1.1 by 1.5 to 2.0 u. 

Sucrose nitrate agar: Substrate growth 
white, becoming straw-colored. Aerial my- 
celium abundant, white, tinged with cream 
to olive-gray. No soluble pigment. 

Glucose -asparagine = agar: 
growth pale yellow. Aerial mycelium white, 
becoming gray. No soluble pigment. 


Substrate 


Glucose-peptone agar: Growth moderate, 
yellow. Aerial mycelium grayish-white. No 
soluble pigment. 

Starch agar: Growth heavy, cream- to 
straw-colored. Aerial mycelium white. 

Potato: Growth slow, secant, white, later 
becoming heavy, gray. Aerial mycelium 
gray. Soluble pigment dark brown. 

Gelatin: Cream-colored, flaky sediment. 
Rapid liquefaction. No soluble pigment. 

Milk: Slow coagulation and peptonization. 
Slight acidification, 


DESCRIPTION OF 


Cellulose: No growth. 

Carbon utilization: No gas from adonitol, 
arabinose, cellobiose, dextrin, dextrose, 
galactose, lactose, levulose, maltose, manni- 
tol, mannose, raffinose, rhamnose, salicin, 
sucrose, or xylose. 

Antagonistic properties: Produces anti- 
biotie novobiocin. 

Habitat: Soil. 

femarks: According to Kuroya et al. 
(1958), this organism is related if not identi- 


cal to S. griseoflavus. 


228. Streptomyces spiralis (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, S. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight or 
spiral-shaped. Spores cylindrical, 1.0 to 1.7 
by 0.9 uw (Fig. 46). 

Sucrose nitrate agar: Growth rough or 
granular, yellowish-golden. Aerial mycelium 
vinaceous-buff to dark grayish-olive. Soluble 
pigment pale vinaceous to fawn-colored. 

Potato: Growth poor, wrinkled, grayish- 
vinaceous. Aerial mycelium white to grayish- 
vinaceous. Plug colored brown around and 
below growth. 

Gelatin: Growth limited, gray. Aerial my- 
celium scant, white. Liquefaction rapid. 
Melanin-negative. 

Milk: Surface growth good. Aerial myce- 
lium abundant, white. Coagulation and 
rapid peptonization. 

Starch: No hydrolysis. 

Nitrate reduction: None. 

Tyrosinase reaction: Negative. 

Habitat: Potato seab. 

Remarks: Krassilnikov (1949) considered 
this organism as belonging to the A. scabies 
group. 


229. Streptomyces sptroverticillatus Shin- 
obu, 1958 (Shinobu, R. Botan. Mag. Tokyo 
71: 87-93, 1958). 

Morphology: Verticil formation usually 
occurs near base of aerial mycelium, but 
generally not so remarkable as in the other 


SPECIES OF STREPTOMYCES 2 


~] 
a | 


FIGURE 46. Sporophores of S. spiralis, showing 
that not all are transformed into spores, X 8,000 
Milan, 


(Courtesy of IE. Baldacci, University of 


Italy). 


verticil-forming species. Occasionally, very 
few tufts on the skirt of the colony. Nitella 

type verticils, generally primary only, seldom 
About 2 to 4 radial 
branches. On synthetic media, many spirals 


secondary. short 


in form of curled tips with | to2 turns, seldom 


D 


3 turns; diameter of spirals about 5 to 8 yu; 


sometimes snail-like and hook-like curls. 


Occasionally loose or closed spirals with 2 to 
3 verticil turns, sinistrorse. Spores spheroid, 
somewhat ellipsoid; about 0.8 uw in length 
(Fig. 47). 


Sucrose nitrate agar: Growth colorless to 


Ficure 47. S. spiroverticillatus (Reproduced 
from: Shinobu, R. Botan. Mag. (Tokyo) 71: 88, 
1958). 


pale brown to yellowish-orange. Aerial my- 

celium thin, somewhat cottony, white. 
Glucose-asparagine Growth pale 

yellow-orange. Aerial mycelium good, cot- 


agar: 


tony, white to brownish. 

Calcium malate agar: Growth yellow- 
orange to light brown. Aerial mycelium 
abundant, cottony, white. 

Nutrient agar: Growth golden yellow to 
buff. No aerial mycelium. Soluble pigment 
pale brown. 

Potato: Growth yellowish-brown to brown. 
Aerial mycelium abundant, cottony, white 
to brownish-white. Soluble pigment brown. 

Milk: Growth yellow to yellow-orange. 
Aerial mycelium poor, light brownish-gray. 
Soluble pigment yellowish-orange. No co- 
agulation; rapid peptonization, 

Gelatin: Growth poor; liquefaction strong. 

Tyrosinase reaction: Somewhat unstable, 
generally positive, weak. 

Diastase reaction: Fairly strong. 

Nitrate reduction: Positive. 

Carbon utilization: Utilizes lactose, fruc- 


THE ACTINOMYCETES, Vol. II 


tose, and xylose. Sucrose and inositol un- 
certain. Does not utilize rhamnose, man- 
nitol, and raffinose. 

Habitat: Soil. 


230. Streptomyces sulphureus (Rivolta, 1882 
emend. Gasperini, 1894) Waksman (Rivolta, 
S. Arch. path. Anat. Phys. 88: 389, 1882; 
Gasperini, G. Centr. Bakt. Abt. 1, 15: 684, 
1894; Waksman, 8S. A. Soil Sci. 8: 102-104, 
1919). 

Synonym: Actinomyces 
Waksman, 1919. 

Not A. sulphureus Berestnew, 1897. 

This organism is usually found in culture 
collections under the name of Actinomyces 
bovis. Baldacci (1937, 1947) emphasized the 
synonymy of this organism, listing as many 
as 13 different names. The following de- 
scription is based upon the data of Waks- 
man (1919), who also spoke of it as A. bovis. 

Sucrose nitrate agar: Growth white, turn- 
ing yellow. Aerial mycelium light, powdery, 
sulfur-yellow. No soluble pigment. 


(Harz) 


bovis 


Calcium malate-glycerol agar: Growth 
brownish. No aerial mycelium. 
Nutrient agar: Growth at first cream- 


colored, later becoming fawn-colored, brown, 
then almost black. Aerial mycelium pale 
yellow-green. No soluble pigment. Melanin- 
negative. 

Glucose agar: Growth yellowish, later be- 
coming dark. Aerial mycelium thin, sulfur- 
yellow. 

Starch: Fair hydrolysis. 

Potato: Growth abundant, much wrin- 
kled, gray to canary-yellow. Aerial myce- 
lium yellow, turning sulfur-yellow. Plug at 
first not pigmented, later turning brownish. 

Gelatin: Growth gray to brownish. No 
aerial mycelium. No pigment. 
Liquefaction rapid at 37°C; slow at 18°C. 

Milk: Surface growth thin, yellowish. 
Coagulation and peptonization. 

Carbon utilization: Ready utilization of 


soluble 


glucose, lactose, sucrose, maltose, glycerol, 
and various organic acids. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 279 


Nitrate reduction: Positive. 

Production of HS: Negative. 

Invertase: None reported. 

Remarks: Ettlinger et al. (1958) considered 
certain strains of this organism as belonging 
to the S. griseus series. 


231. Streptomyces tanashiensis Hata et al., 
1952 (Hata, T., Ohki, N., and Higuchi, T. 
J. Antibiotics (Japan) 5: 529-534, 1952). 

Morphology: Sporophores almost straight. 
Spores spherical to oval, 1.0 by 1.2 yp. 

Sucrose nitrate agar: Growth grayish- 
yellow. Aerial mycelium white-gray, turning 
brownish-gray. Soluble pigment light yellow. 

Potato: Growth brown. Aerial mycelium 
dark gray to whitish-gray. Soluble pigment 
dark brown. 

Gelatin: Soluble pigment brown. Rapid 
liquefaction. 

Milk: Yellowish surface ring. Coagulation 
and peptonization. 

Starch: Hydrolysis. Most suitable for anti- 
biotic production. 

Nitrate reduction: Negative. 

Tyrosinase reaction: Positive. 

Production of H.S: Positive. 

Optimum pH: 5.8 to 6.5. 

Antagonistic properties: Produces luteo- 
mycin. 

Habitat: Soil. 

Remarks: 


antibioticus. 


Resembles S. aureus and S. 


232. Streptomyces tendae Ettlinger et al., 
1958 (Ettlinger, L., Corbaz, R., and Hitter, 
R. Arch. Mikrobiol. 31: 351, 1958). 

Morphology: Sporophores form verticils; 
chains of spores as open, regular spirals. 
Spores smooth (Pl. I ¢). 

Glycerol nitrate agar: Growth thin, light 
yellow. No aerial mycelium. No soluble pig- 
ment. 

Glucose-asparagine agar: Growth lhght 
yellow to light carmine. Aerial mycelium 
cottony, clnnamon-brown. 

Calcium malate agar: Growth light yellow 


to brownish-yellow. Soluble pigment brown- 
ish-yellow. 

Gelatin: Growth lght yellow. Aerial my- 
celium sparse. Liquefaction limited. Soluble 
pigment dark brown. 

Starch agar: Growth thin, light yellow. 
Limited hydrolysis of starch. 

Potato: to dark. Aerial 
mycelium powdery, chalk-white. 

Milk: Growth brownish-yellow. Aerial my- 


Growth brown 


celium sparse. No coagulation; weak pep- 
tonization. 

Antagonistic properties: Produces anti- 
biotic carbomycin. 

Habitat: Soils in France. 

Remarks: Organism said to be melanin- 
negative, although dark brown pigment re- 
ported on gelatin. 


233. Streptomyces tenuis (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight. Spores 
cylindrical, 0.9 by 0.8 u. 

Sucrose nitrate agar: Growth thin, flat, 
yellowish-drab. Aerial mycelium deep olive- 
buff. Soluble pigment pale orange-yellow. 

Glucose agar: Growth thin, flat. Aerial 
mycelium olive-buff. Soluble pigment green. 

Potato: Growth good. Aerial mycelium 
deep olive-buff. Soluble pigment gray to 
olive to black. 

Nutrient potato agar: Growth wrinkled, 
grayish. Aerial mycelium white to vinaceous- 
fawn. Soluble pigment golden brown. 

Gelatin: Growth pale gray. Aerial myce- 
lium scant, white. Rapid hquefaction. Solu- 
ble pigment yellow. 

Milk: Growth Aerial 
white. Coagulation; incomplete peptoniza- 
tion. 

Starch: Hydrolysis. 

Nitrate reduction: Negative. 


good. mycelium 


Tyrosinase reaction: Negative. 
Habitat: Potato scab. 


234. Streptomyces thioluteus Okami, 1952 


280 


(Okami, Y. Taxonomic study of antibiotic 
streptomyces. Thesis, Hokkaido University, 
Japan, 1952). 

Morphology: Aerial hyphae with few 
branches. No spirals, but verticils produced 
oceasionally, depending on composition of 
medium. 

Glycerol nitrate agar: Growth yellowish- 
brown, penetrates deep into medium. Aerial 
mycelium thin, white, with dark yellowish 
tinge. Soluble pigment yellowish-brown. 

Nutrient agar: Growth wrinkled, yellow- 
ish-brown. Aerial mycelium scant, white. 
Soluble pigment slight, yellowish-brown. 

Starch agar: Growth thin, cream-colored. 
No aerial mycelium. No hydrolysis. 

Gelatin: Growth yellowish-brown at bot- 
tom of liquefied portion. No aerial mycelium. 
Soluble pigment slght, yellowish-brown. 
Slow liquefaction. 

Potato: Growth wrinkled, cream to yel- 
lowish. No aerial mycelium. Soluble pigment 
slight, yellowish-brown. 

Milk: Growth on surface of milk yellowish. 
Aerial mycelium scant. Coagulation occurs 
in 2 to 3 days, followed by slow peptoniza- 
tion. 

Blood agar: Growth dark gray with green- 
ish tinge. Aerial mycelium dark. No hemoly- 
sis. 

Nitrate reduction: Negative. 

Production of H.S: Negative. 

Antagonistic properties: Produces anti- 


fungal substance, aureothricin (Maeda, 
1953). 

Type culture: ATCC 12,310. 

235. Streptomyces tumuli (Millard and 


Beeley, 1927) Waksman (Millard, W. A. 
and Beeley, fF. Ann. Appl. Biol. 14: 296-311, 
1927). 

Sucrose nitrate agar: Growth gray, later 
becoming opaque dark. Aerial mycelium 
arises at center of growth, at first white, 
later becoming pale gray. Surface of growth 
covered with colorless drops leaving small 
black craters. No soluble pigment. 


THE ACTINOMYCETES, Vol. II 


Glucose-asparagine agar: Growth wrin- 
kled, pale gray. Aerial mycelium white, 
arising in concentric rings around a dark 
bare center. Soluble pigment olive-colored. 

Nutrient agar: Growth good, lustrous, 
slimy, gray. No aerial mycelium. No soluble 
pigment. Melanin-negative. 

Potato: Growth heavy, slimy, black. No 
aerial mycelium. Soluble pigment grayish- 
brown. 

Gelatin: Growth beaded. No aerial myce- 
hum. Liquefaction rapid. No soluble pig- 
ment. 

Milk: Growth good; no aerial mycelium. 
Coagulation and slight peptonization. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Habitat: Mound seab of mangels. 


236. Streptomyces venezuelae Ehrlich et al., 
1948 (Ehrlich, J., Gottheb, D., Burkholder, 
P. R., Anderson, L. E., and Pridham, T. G. 
J. Bacteriol. 56: 467-477, 1948; Pridham, 
T. G. and Gottheb,. D. J. Bactenol:*56: 
107-114, 1948). 

Morphology: Sporophores monopodially 
branched, straight or shghtly and irregularly 
curved. Spores oval to oblong, 0.4 to 0.9 by 
0.7 to 1.6 «, smooth. (PE dlp): 

Sucrose nitrate agar: Aerial mycelum 
light lavender. 

Nutrient agar: Substrate growth yellow 
to brown. Aerial mycelium gray. Soluble 
pigment dark brown to black. 

Calcium malate agar: Substrate growth 
vellow to brown; aerial mycelium gray. 
Soluble pigment dark 


Glucose agar: 


brown. 

Potato: Growth abundant, gray to dark 
brown. Aerial mycelium gray. Soluble pig- 
ment dark brown or black. 

Gelatin: Liquefaction rapid. Soluble pig- 
ment dark brown. 

Milk: Peptonization. 
dark brown. 

Starch agar: Growth white to lavender. 


Soluble pigment 


Hydrolysis. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Nitrate reduction: Positive. 

Tyrosinase reaction: Positive. 

Production of HS: Positive. 

Carbon utilization: Good growth: xylose, 
arabinose, rhamnose, D-glucose, D-mannose, 
p-fructose, bD-galactose, cellobiose, starch, 
dextrin, glycerol, acetate, citrate, succinate, 
and salicin. Shght or no growth: b-ribose, 
sucrose, raffinose, inulin, erythritol, dulcitol, 
mannitol, sorbitol, inositol, and malate. No 
growth: formate, oxalate, tartrate, salicylate, 
phenol, 0-cresol, m-cresol, p-cresol. 

Antagonistic properties: Produces chlor- 
amphenicol, an antibiotic active against 
various gram-positive and gram-negative 
bacteria, rickettsiae, and psittacosis group. 

Habitat: Different soils. 

Remarks: This organism is variable, re- 
sembling in some respects S. lavendulae, al- 
though Okami (1956) found it to be mark- 
edly different. Krassilnikov described a form 
under the name A. rectus, and a related form, 
A. rectus brunneus, which belong to this 
group. Gause et al. (1957) described a form 
as A. venezuelae var. sprralis. S. phaeochromo- 
genes var. chloromyceticus Okami is identical 
with S. venezuelae. 

Morais et al. (1958) described a variety of 
S. venezuelae as roseosport with a_rose- 
colored rather than lavender aerial myce- 
lium, not producing any antibiotic and not 
chomogenic on organic media. 

Type species: IMRU 3534; ATCC 10,712. 

237. Streptomyces verne (Waksman and 
Curtis, 1916) Waksman and Henrici (Waks- 
man, 8S. A. and Curtis, R. E. Soil Sci. 1: 120, 
1916; 8: 156, 1919). 

Morphology: According to Jensen (1931), 
sporophores are long, spiral-shaped. Spores 
spherical and oval. 

Sucrose nitrate agar: Growth abundant, 
spreading, lichenoid, glossy, yellowish, be- 
coming brownish. Capacity to produce aerial 
mycelium lost on cultivation. 

Glucose-asparagine agar: Growth abun- 
dant, lichenoid, center raised, gray with 


281 


purplish tinge. No aerial mycelium. Soluble 
pigment faint brown. 

Nutrient agar: Colonies small, grayish, 
with depressed center, becoming wrinkled. 
No aerial mycelium. No soluble pigment. 

Potato: Growth wrinkled, cream-colored, 
becoming gray. Aerial mycelium absent or 
scant, white. 

Gelatin: Colonies small, cream-colored. 
No aerial mycelium. Soluble pigment green, 
a property lost on continued cultivation. 
Rapid liquefaction. 

Milk: Ring pinkish-brown. Coagulation 
and rapid peptonization. 

Starch: Growth scant, restricted, brown- 
ish; hydrolysis rapid. 

Cellulose: Growth good. 

Nitrate reduction: Positive. 

Production of HS: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Limited activity 
against some bacteria. 

Remarks: Soluble green pigment produced 
by freshly isolated cultures; in time, this pig- 
ment becomes brown. According to Ettlin- 
ger et al. (1958), this organism should be 
regarded as a strain of S. olivaceus. 

Type Culture: IMRU 3353. 

238. Streptomyces — verticillatus — (KXriss, 
1938) Waksman (Ixriss, A. Mikrobiologiya 7: 
LQ5—111, 1938). 

Morphology: Substrate mycelium pro- 
duced by monopodial branching. Aerial my- 
celium characterized by primary verticils 
produced on straight The 
number of verticils at the proximal ends of 
the primary sterile hyphae is much larger 
than in the younger portions. Secondary 
verticils are also produced at the ends of the 
primary. Spores cylindrical and oblong, 1.0 
to 1.7 by 0.8 p. 

Sucrose nitrate agar: Aerial mycelium well 
developed, velvety, at first white, later dark 


sporophores. 


gray or gray-green. 
Nutrient agar: Growth brown. No aerial 
mycelium. Soluble pigment brown. 


282 


Potato: Soluble pigment brown. 

Gelatin: Rapid liquefaction. 

Milk: Coagulation and peptonization. 

Starch: Hydrolysis. 

Cellulose: No growth. 

Nitrate reduction: Rapid. 

Sucrose: Inversion. 

Production of H.S: Positive. 

Antagonistic properties: Weak. 

Habitat: Rhizosphere of wheat grown in a 
salinized soil. 

Remarks: A. verticillatus viridans was de- 
scribed by Krassilnikov (1941) as a substrain 
of this organism. 


239. Streptomyces violaceoniger (Waksman 
and Curtis, 1916) Waksman and Henrici, 
1948 (Waksman, 8. A. and Curtis, R. E. 
Soil Sei. 1: 111, 1916). 

Synonym: S. violaceus-niger. 

Morphology: The sporogenous hyphae are 
frequently sterile. Sporophores monopodially 
branched. Waksman and Curtis (1916) re- 
ported no spirals, but Ettlinger et al. (1958) 
found compact spirals. Spores spherical and 
oval, 1:2°to 1.5 by 1.2 to 2.34, Smooth (PI. 
TL a); 

Sucrose nitrate agar: Growth at first dark 
eray, turning almost black. Aerial mycelium 
white to gray after the colony is well de- 
veloped. Soluble pigment at first bluish, later 
turning almost black. 

Potato: Growth at first very shght, but 
after 48 hours develops into continuous, 
thick yellowish-gray smear, which later 
turns brown, with white aerial mycelium 
covering the growth. Melanin-negative. 

Gelatin: Growth gray; no aerial mycelium. 
Liquefaction rapid. No change in color. 

Production of H.oS: Negative. 

Antagonistic properties: Produces anti- 
biotic nigericin, 

Habitat: Soil. 

temarks: According to Ettlinger et al. 
(1948) the color of the aerial mycelium is 
carmine-red to cinnamon-brown; with age, 
the aerial mycelium liquefies and turns black. 


THE ACTINOMYCETES, Vol. II 


This organism was believed to belong to the 
S. hygroscopicus group. Nomi (1960) came 
to similar conclusions. 


240. Streptomyces violaceoruber (Waks- 
man and Curtis, 1916) Waksman (Waksman, 
S. A. and Curtis, R.' E. Soil Sci. 12 110=111, 
1916; 8: 160-163, 1919). 

This organism has an interesting history. 
In the original description of Waksman and 
Curtis (1916), it was listed in the text (p. 
110) as A. violaceus, the word “ruber” being 
left out due to poor proof-reading; in the 
key, however (p. 130), as well as in the 
following paper by Waksman (1919), in 
which a complete description was given, it was 
correctly listed as A. violaceus-ruber. The 
above error was unfortunately repeated in 
the first and second (p. 3874) editions of 
Bergey’s Manual. In the third edition of this 
manual (1930), Bergey himself changed the 
name of this organism to Actinomyces Waks- 
mani (p. 489). In the fourth and fifth (p. 
867) editions, it was changed to Actinomyces 
coelicolor (Miller) Lieske, and finally in the 
sixth and seventh editions to Streptomyces 
coelicolor (Miller) Waksman and Henrici. 
Only the recent studies in which both or- 
ganisms, S. coelicolor Miller and S. violaceo- 
ruber Waksman and Curtis, were directly 


compared (Kutzner, 1956; Zihner and 
Ettlinger, 1957; Kutzner and Waksman, 


1959) demonstrated that they are distinctly 
different. 

There are marked physiological and bio- 
chemical differences between SS. coelicolor 
and S. violaceoruber. They particularly in- 
clude differences in color and morphology of 
the aerial mycelium, antagonistic properties, 
and pigment production. S. coelicolor is ac- 
tive upon fungi and yeasts, as first shown by 
Miller; several strains of S. violaceoruber 
produce antibacterial antibiotics, such as 
actinorhodin, coelicolorin, and mycetin. The 
pigment of S. coelicolor changes to green at 
an alkaline reaction, that of S. violaceoruber 
to blue. The nature of the pigment has been 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


studied by Conn (1943) and by Cochrane 
and Conn (1947). 

Various strains closely related to S. vio- 
laceoruber have been isolated all over the 
world; some have been listed as varieties, 
such as achrous and flavus (Gause et al., 
1957). Most of the strains now in the culture 
collections, designated as S. coelicolor, ac- 
tually belong to S. violaceoruber. 

Type culture: Waksman and Curtis strain 
No. 3030, available in the IMRU culture 
collection. 

Synonyms: 

A. violaceus Waksman and 
(Waksman and Curtis, 1916). 

A. violaceus-ruber Waksman and Curtis 
(Waksman, 1918). 

A. waksmani Bergey (Bergey’s Man- 
ual, 3rd ed. 1930). 

A. coelicolor (Miller) Lieske (Bergey’s 
Manual, 4th and 5th ed., 1934, 1939). 

S. coelicolor (Miller) Waksman 
Henrici (Bergey’s Manual 6th 
7th ed., 1948, 1957). 

A. coelicolor (Miiller) 
(KXrassilnikov, 1941). 

A. coelicolor WKrassilnikov (Gause et al., 
1957). 

Possible synonym: A. 
new emend. Krassilnikoy. 

Morphology: Aerial mycelium  mono- 
podially branched; abundant formation of 
spirals with 3 to 8 turns, sinistrorse. Ac- 
cording to Naganishi and Nomi (1954), two 
or more sporulating branches may grow from 
the same spot on the main sporophore. 
Secondary branches may also be produced. 
Terminal branches are often arranged in 
clusters or umbellate forms. Terminal hy- 
phae carry many spirals. Spores spherical to 
oval, 0.7 to 1.0 by 0.8 to 1.5 » (PI. IV Ab). 
Surface of spores smooth. Asporogenous, 
nonpigmented strains can be obtained by 
plating out cultures on carbohydrate-free 
synthetic media containing nontoxic surface- 
acting agents (Erikson, 1955b). 


Curtis 


and 
and 


Kkrassilnikov 


pluricolor Berest- 


283 


Sucrose nitrate agar: Substrate growth 
colorless at first, becoming red, 
to dark blue. Aerial mycelium 


thin, powdery, white, becoming ash-gray, 


abundant, 
then blue 
with a bluish tinge; on some media, light 
pink to cinnamon; sometimes blue drops can 
be observed on the surface of the aerial my- 
celium. Soluble red pigment on acid media, 
changing to dark blue as medium becomes 
alkaline. 

Glycerol-asparagine agar: Growth good, 
violet to deep blue. Soluble pigment diffuses 
through medium. 

Glucose-asparagine agar: Growth poor; 
red pigment does not diffuse readily. 

Nutrient agar: Growth white, becoming 
red with white margin. No soluble brown 
pigment. Melanin-negative. 

Potato: Small, brownish, lichenoid colonies. 
Aerial mycelium white. Mycelium and plug 
gradually colored red and blue. 

Gelatin: Growth cream-colored, becoming 
pink or blue. Liquefaction slow. 

Milk: Gray surface ring, with red or blue 
tinge. Coagulation limited; peptonization 
rapid. 

Starch agar: 
rapid. 

Cellulose: Growth good. 

Nitrate reduction: Excellent. 

Sucrose: Inversion. 

Carbon 


Growth pink. Hydrolysis 


sources: Utilizes L-xylose, L- 
arabinose, L-rhamnose, b-fructose, raffinose 
(some strains only faintly), pb-mannitol. None 
or poor utilization by most strains: sucrose, 
Inulin. 

Antagonistic properties: Most strains do 
not show any strong antagonistic effect; 
several cultures, which seem to belong or are 
closely related to S. violaceoruber, produce 
coelicolorin, actinorhodin, 
and mycetin. 


streptocyanin, 


Habitat: Very common, especially in field 
soils. 

temarks: Ettlinger ef al. (1958) considered 
S. violaceoruber, 


quite incorrectly, as a 


234 


strain of S. fradiae. Krassilnikov (1949) 
considered it as a synonym of S. coelzcolor. 


Type culture: IMRU 3030. 


241. Streptomyces violaceus (Gasperini, 
1894, emend. KKrassilnikov) Waksman (Gas- 
perini, G. Centr. Bakteriol. Parasitenk., 
Abt. I 15: 684, 1894; Krassilnikov, N. A. 
Actinomycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 15, 1941). 

Morphology: Aerial hyphae long, straight, 
seldom 


also branched 


short, 


branching; 


THE ACTINOMYCETES, Vol. II 


hyphae. Sporophores produce open, sinis- 
trorse spirals with 2 to 3 coils. Spores spheri- 
cal and oval (Fig. 48). 

Agar media: Substrate growth lichenoid, 
at first red, red-blue, 
purple-violet. Some cultures produce fat 


becoming finally 
droplets in the colony, pigmented red or 
purple. Aerial mycelium white to gray, pro- 
duced poorly or not at all; some substrates, 
like cellulose, paraffin, or fats, favor forma- 
tion of aerial mycelium. Different pigments 


Ficure 48. Sporophores and spores of S. violaceus, grown for 11 days on glucose-asparagine agar 


(top, hyphae); for 8 days on yeast-glucose agar (bottom left, sporophore); for 4 days on potato agar 
(bottom right, spores) X 18,500 (Reproduced from: Lechevalier, H. A. and Tikhonienko, A. 8. Mikro- 


biologiya 29: 43-50, 1960). 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


are formed in different media and under dit- 
ferent conditions of growth. Pigments dis- 
medium do with 


solved in not 


reaction. 


change 


Sucrose nitrate agar: Growth dark brown. 
Aerial mycelium white. Soluble pigment be- 
comes violet to dark violet. 

Potato: Growth red-brown to 
Aerial mycelium, if present, white. Soluble 
pigment grayish-brown. 

Gelatin: Aerial mycelium white. Liquetac- 
tion slow. Soluble pigment gray-brown. 

Milk: Growth-grayish brown. Coagulation 
questionable; peptonization slow. 

Starch: Hydrolysis weak. 

Cellulose: Growth weak, 
mycelium light gray. 

Nitrate reduction: Positive. 

Sucrose: Rapid inversion. 

Pigment: According to Kriss (1936), the 
pigment is soluble in water and in 96 per 
cent alcohol. 

Melanin: According to Hoffmann (1958), 
this species is melanin-positive. 

Antagonistic properties: Exerts strong 
antagonistic effect upon various gram-posi- 
tive bacteria. 

Remarks: According to Krassilnikoyv, this 
species includes A. violacea Rossi-Doria, A. 
violaceus-caesart. Waksman and Curtis, Ac- 
tinomyces 103 and 109 Lieske; also A. 
incanescens Wollenweber and A. brasiliensis 
Lindenberg (the last is probably a Nocardia). 
A subspecies, A. violaceus chromogenes is also 
included. Some of the cultures described by 
Gause ef al. (1957) may also be included 
here, such as A. lateritius, A. roseoviolaceus, 
A. violaceorectus, A. viridoviolaceus, and A. 
violaceus var. rubescens. 

Type culture: IMRU 3497. 


brown. 


violet. Aerial 


242. Streptomyces virgatus (Krassilnikov) 


Waksman (Krassilnikov, N. A. Actino- 
mycetales. Izvest. Akad. Nauk, SSSR, 


Moskau, p. 32, 1941). 
Morphology: Sporophores short, in form 


289 
of tufts. Spirals produced rarely. Spores 
cylindrical, elongated; in some strains round 
to oval. 

Agar media: Substrate growth yellow- 
green to citron-yellow or pure yellow; on 
some media pale green. Pigment insoluble. 
Some strains produce a brown substance in 
protein media. Aerial mycelium weakly de- 
veloped, white or pale yellow. 

Gelatin: Liquefaction rapid. 

Milk: 
rapid. 

Starch: Hydrolysis rapid. 

Cellulose: No growth. 


Coagulation and  peptonization 


Sucrose: Inversion. 

Nitrate reduction: Positive. 
Antagonistic properties: None. 
Habitat: Soil. 


243. Streptomyces virginiae Grundy et al., 
1952 (Grundy, W. E., Whitman, A. L., 
Rdzok, E. J., Hanes, M. E., and Sylvester, 
J. C. Antibiotics & Chemotherapy 2: 399- 
408, 1952). 

Morphology: usually 
straight; occasionally spirals are observed at 


Sporophores 


or near the tips of the hyphae. Spores cylin- 
drical deto Wo byOs7o toweO a: 
nitrate Growth 
cream-colored. Aerial mycelium light gray- 
ish-lavender. No soluble pigment. 
Glucose-asparagine agar: Growth sparse, 
cream-colored to light brown. No aerial my- 


Sucrose agar: sparse, 


cellum. 

Calcium malate agar: Growth abundant, 
cream-colored. Aerial mycelium white, be- 
coming tinged with grayish-pink to lavender. 

Nutrient Growth white, 
turning cream-colored to light brown. Aerial 
mycelium white, turning light grayish-pink 
to lavender. Soluble pigment light brown. 

Oatmeal agar: Growth abundant, cream- 
colored, turning golden brown. Aerial my- 
celium abundant, light rose, turning lavender 
and gray. Soluble pigment pale yellow, turn- 


agar: sparse, 


ing light brown. 


286 


Starch agar: Growth thin, colorless. Aerial 


mycelium rose to lavender-colored. Hy- 
drolysis. 
Potato: Growth abundant, spreading, 


brownish. Aerial mycelium grayish-pink to 
lavender. Browning of the potato. 

Gelatin: Surface pellicle gray to brownish. 
Aerial mycelium thin, white. Soluble pig- 
ment brown. Liquefaction slow. 

Milk: Growth brown. Coagulation none; 
peptonization slow. Milk becomes dark gray- 
brown or black. 

Nitrate reduction: Limited or absent. 

Production of H.S: Positive. 

Carbon utilization: Utilizes glucose, man- 
nose, galactose, maltose, starch, glycerol, 
sodium acetate, sodium citrate. Does not 
utilize xylose, lactose, sucrose, mannitol, 
sorbitol, potassium sodium tartrate. 

Antagonistic properties: Produces an 
antibiotic, actithiazie acid. 

Remarks: Various related organisms have 
been listed. It is sufficient to mention 4A. 
gobitricinit, A. roseolus, A. syringini, and A. 
roseolilacinus, described by Gause et al. 
(1957). 

Type culture: IMRU 3651. 


244. Streptomyces viridans (Krassilnikovy, 
1941) (Krassilnikov, N. A. Actinomycetales. 
Izvest. Akad. Nauk. SSSR, Moskau, 1941). 

Morphology: Sporophores branched, spi- 
ral-shaped. Spores cylindrical. 

Glycerol nitrate agar: Growth olive-green 
with soluble green pigment. Aerial mycelium 
dark gray, olive-colored, or gray-green, vel- 
vety, covering the whole growth. 

Nutrient agar: Growth brown-green. Sol- 
uble pigment brownish. Melanin-negative. 

Potato: Growth brown. Aerial mycelum 
light gray. Soluble pigment olive-green 
(Hoffmann, 1958). 

Gelatin: Rapid liquefaction. 

Milk: No coagulation; rapid peptoniza- 
tion; soluble brown pigment. 

Starch: Hydrolysis rapid. 

Cellulose: Growth poor. 


THE ACTINOMYCETES, Vol. II 


Nitrate: Reduction to nitrite. 
Sucrose: Inversion rapid. 


Antagonistic properties: None; some 
strains are weakly active. 

femarks: Related to S. intermedius. 
Drechsler (1919) described two similar 


strains, Nos. X and XIV. Gause et al. 
(1957) described a related strain as A. roseo- 
viridis. 

245. Streptomyces viridis (Lombardo-Pel- 
legrino, 1903) Waksman (Lombardo-Pelle- 
grino, P. Riforma med. 39: 1065-1067, 1903. 
Summarized by Baldacci, E. Atti ist. botan. 
“Giovanni Briosi” e lab. crittogam. univ. 
Pavia (Ser. IV) 11: 221-223, 1939). 

Morphology: Sporophores long or short, 
straight, undulated; frequently producing 
broom-shaped clumps. Spores ovoid, 0.7 to 
1.4 w in diameter. 

Agar media: Substrate growth on all media 
at first hyaline, later turning green to dark 
green. Soluble pigment green. The cultures 
also grow under anaerobic conditions, but 
produce no soluble pigment. Aerial mycelium 
on all media cottony, whitish to grayish. 

Potato: Growth dark violet. Aerial my- 
celium white. Melanin-negative. 

Production of H.S: Positive. 

Antagonistic properties: Not reported, or 
negative. 

Habitat: Soil. 

temarks: Baldacci and Comaschi (1956) 
concluded that the culture described by 
Krainsky (1914) as A. griseus belongs more 
accurately to the S. viridis series. According 
to Hoffmann (1958), the A. griseus Krainsky 
appears to belong to this group, although he 
refers to it as A. griseus Krassilnikoy. It is 
said to produce broom-shaped sporophores 
with spirals. Growth colorless, turning light 
brown. Aerial mycelium velvety, light gray 
turning dark dray. No soluble pigment. 
Melanin-negative. Growth on potato lichen- 
oid. Milk not coagulated, but peptonized. 
Gelatin liquefied. Starch hydrolyzed. It 
grows on cellulose. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


Certain other forms belonging to this 
eroup have been described, such as A. griseus 
variabilis and A. griseus zonatus of Krassil- 
nikov (1949), A. badius and A. 
(1957). 


listed viridis sterilis as a strain that lost the 


malachiticus 


of Gause et al. Krassilnikov also 


capacity to produce aerial mycelium. 


Millard and Burr (1926) described, under 


A. viridis, an organism that produces dark 
to black growth on sucrose-nitrate agar, 
with a mouse-gray aerial mycelium, gradu- 
ally black. On 
growth is at first colorless, gradually becom- 


becoming nutrient agar, 
ing gray; aerial mycelium gray to mouse- 
gray. On gelatin, it produces a thin colorless 
growth and a faint brownish pigment; rapid 
liquefaction. 

Duché (1934) described an organism under 
the name A. viridis; he later changed this 
name to A. baarnensis. This organism was 
isolated as a contaminant of cultures of S. 


albus and S. lavendulae. 


IMRU 


Type culture: 
Millard and Burr). 


Sate (Strain: OL 


237 


246. Streptomyces viridochromogenes (Kra- 
1948 
(Xrainsky, A. Centr. Bakteriol. Parasitenk. 
Abt. II., 41: 684-685, 1914). 

Morphology: Sporophores monopodially 


insky, 1914) Waksman and Henrici., 


branched, with numerous open or compact, 
sinistrorse spirals, 3 to 5 p in diameter, oc- 
curring as side branches. Spores short, oval 
or spherical, 1.25 to 1.5 w (Figs. 49-51); sur- 
face covered with long spines (Pl. II k). 

Sucrose nitrate agar: Growth cream-col- 
ored with dark center, becoming dark green; 
reverse yellowish to light cadmium. Aerial 
mycelium white, becoming light green to 
light blue. 

Glucose-asparagine agar: Growth abund- 
ant, spreading, wrinkled, gray, becoming 
black. Aerial mycelium appears late; white, 
later becoming green to light blue. 

Nutrient agar: Growth restricted, gray, 
with greenish tinge. No aerial mycelium. 
Soluble pigment brown. 

Potato: Growth abundant, gray-brown. 


Figure 49. Chains of spores of S. viridochromogenes, grown for 16 days on glucose-asparagine-CaCl» 


agar, X 13,500 (Reproduced from: Lechevalier, H. . 


1960). 


\. and Tikhonienko, A. 8S. Mikrobiologiya 29: 43-50, 


233 THE ACTINOMYCETES, Vol. II 


Figure 50. S. viridochromogenes grown for 380 days on potato agar, X 13,500 (Reproduced from: 


Lechevalier, H. 


Aerial mycelium white. 
black. 


Gelatin: Surface growth cream-colored, 


Soluble pigment 


becoming greenish. Positive liquefaction. 
Soluble pigment brown. 

Milk: Surface growth dark brown; coagu- 
lation and peptonization. 

Starch agar: Colonies circular, spreading, 
yellowish. Hydrolysis. 

Cellulose: No growth. 

Sucrose inversion: Positive. 

Nitrate reduction: Positive. 

Production of Hos: Positive. 

Tyrosinase reaction: Positive. 

Temperature: Optimum 37°C, 

Antagonistic properties: Active upon 
fungi. 


Habitat: Soil. 


A. and Tikhonienko, A. S. Mikrobiologiya 29: 43-50, 1960). 


Remarks: This group occurs abundantly 
(1958) 
various forms under different names, such 


in nature. Gause et al. described 


as A. bicolor, A. coeruleofuscus, A. coeruleo- 


rubidus, and A. coerulescens, and a variety, 
longisporus. According to Ettlinger et al. 
(1958), S. chartreusis also belongs to this 
group. 

247. 


Taber, 


Waksman 
(Waksman, S. A. 
A guide to the classification 


Streptomyces viridoflavus 
1953 


Lechevalhier, H. 


and and 
and identification of the actinomycetes and 
their antibiotics. The Williams & Wilkins 
Co., Baltimore, 1953, p. 66). 

Not A. viridiflavus Duché. 

Morphology: Sporophores formed in fas- 
cicles; tufts, with some curling of tips, are 
produced on certain media. Tendency to lose 


DESCRIPTION OF 


SPECIES OF STREPTOMYCES 


289 


FIGuRE 51. 
Milan, Italy). 


property of producing aerial mycelium. Sub- 

merged sporulating lateral branches form 

single spores at the tips. 

limited, 
Aerial 


Sucrose nitrate Growth 


agar: 


cream-colored to yellowish green. 


Sporophores of S. viridochromogenes, X 30,000 (Courtesy of E. 


Baldacci, University of 


mycelium usually absent. No soluble pig- 
ment. 

Glucose nutrient agar: Growth lichenoid, 
vellowish-brown to olive-brown. Aerial my- 


eelium abundant, later covering the whole 


290 


surface of growth with a mat, yellowish to 
gray. Soluble pigment brownish or absent. 

Glucose-asparagine agar: Growth moist, 
vellow to yellow-green. Aerial mycelium 
abundant, grayish-yellow to sulfur-yellow, 
later overgrown by white sporulating hy- 
phae. Soluble pigment absent or faint yellow. 

Nutrient agar: Growth moist, gray to 
light green with green to almost bluish tinge 
at bottom of slant. Nonsporulating aerial 
mycelium appears much later; it is white to 
gray. No soluble pigment. 

Potato: Growth lchenoid, brownish to 
greenish-yellow to dark olive-green. Aerial 
mycelium absent, or formed as thin, yellow- 
ish layer on drier portions of growth. Soluble 
pigment absent or dark brown. 

Gelatin: Growth in form of surface ring, 
canary-yellow. Slight liquefaction. Soluble 
pigment brown to dark brown, a property 
that may be lost on cultivation. 

Starch: Hydrolysis. 

Cellulose: Limited growth, no destruction 
of cellulose. 

Production of H.S: Negative. 

Carbon utilization: No growth with su- 
crose, lactose, or rhamnose; good growth on 
mannose and glucose. 

Antagonistic properties: Produces an anti- 
fungal substance, candidin. 

Habitat: Soil. 

Type culture: IMRU 3685. 


248. Streptomyces viridogenes (Millard and 
Burr, 1926) Waksman (S. viridis of Millard, 
W. A. and Burr, 8. Ann. Appl. Biol. 13: 
580, 1926). 

Morphology: Sporophores long, sympodi- 
ally branched, straight. Spores spherical, 
0.9 u, smooth (PI. II 0). 

Sucrose nitrate agar: Growth abundant. 
Aerial mycelium olive-gray. Soluble pigment 
greenish-yellow to blackish-green. 

Glucose-asparagine agar: Growth smooth, 
raised, olive-buff. Aerial mycelium abund- 
ant, ight gray to deep mouse-gray. Soluble 
pigment yellowish to greenish-yellow. 


THE ACTINOMYCETES, Vol. II 


Nutrient agar: Growth lichenoid, cream- 
colored. No aerial mycelium. No soluble 
pigment. 

Nutrient agar with glucose: Growth gray 
to black. Aerial mycelium gray. Soluble pig- 
ment dark brown. 

Starch agar: Growth gray to brown. Aerial 
mycelium thin, white. Hydrolysis positive. 

Potato: Growth gray to olive-gray. Aerial 
mycelium either absent or white, turning 
gray. Soluble pigment brown. 

Gelatin: Growth grayish. Aerial mycelium 
scant, white to gray. Liquefaction rapid. 
Soluble pigment light golden brown. 

Milk: Surface growth good. Aerial my- 
celium scant, white. Coagulation rapid and 
peptonization gradual. 

Nitrate reduction: Positive. 

Tyrosinase reaction: Negative. 

Temperature: Grows well at 37.5°C 

Habitat: Potato scab. 

Yemarks: Ettlinger et al. (1958) reported 
that this species is melanin-negative. 


249. Streptomyces wedmorensis (Maillard 
and Burr, 1926) Waksman (Millard, W. A. 
and Burr, 8. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight, 
branched. Spores oblong, 0.8 to 0.9 by 0.6 to 
0.8 yu. 

Sucrose nitrate agar: Growth flat, thin, 
grayish. Aerial mycelium white to gray. 

Nutrient potato agar: Growth wrinkled, 


grayish. No aerial mycelium. Melanin- 
negative. 


Potato: Growth wrinkled, grayish. Aerial 
mycelium white. Plug pigmented drab. 

Gelatin: Growth fair. No aerial mycelium. 
Liquefaction medium. 

Milk: Growth greenish. Coagulation and 
slow peptonization. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Tyrosinase reaction: Negative. 

Temperature: Grows well at 37.5° 

Habitat: Potato tubers. 


DESCRIPTION OF SPECIES OF STREPTOMYCES 


250. Streptomyces willmoret (Erikson 
1935) Waksman and Henrici, 1948. (Erik- 
son, D. Med. Research Council (Brit.) 
Spec. Rept. Ser. No. 203: 19-20, 1935). 

Morphology: Submerged growth 
unicellular mycelium frequently 
branched at short intervals, presenting pe- 
eculiar clubbed and budding forms with 
separate, round, cells. 
The filaments are characteristically long, 
homogeneous, and much interwoven. Aerial 
mycelium profuse in most media, with a 
marked tendency to produce loose spirals 
with chains of ellipsoidal spores. Thick aerial 
clusters may also be formed. 

Glucose-asparagine agar: Growth color- 
less, wrinkled, confluent, with smooth entire 
large discrete colonies like flat 
rosettes. Aerial mycelium scant, white. 


cOon- 


sists of 


occasional swollen 


margin; 


Glycerol nitrate agar: Round, smooth, 
cream-colored colonies, heavy texture, mar- 
gin submerged. Stiff, sparse aerial spikes. 

Peptone-beet extract or nutrient agar: 
Growth heavy, colorless, lichenoid, rounded 
elevation covered with white aerial myce- 
lium. Later, submerged margin, round con- 
fluent growth; aerial mycelium marked in 
concentric zones. 

Potato agar: Fair growth, partly sub- 
merged. Aerial mycelium grayish-white. 

Gelatin: Colonies minute, colorless. Posi- 
tive liquefaction. 

Milk: Coagulation and slow peptonization. 

Dorset’s egg medium: Large, round, color- 
less, scale-like colonies, radially wrinkled, 
later growth brownish; medium discolored. 

Serum agar: Smooth colorless discoid col- 
onies; marked umbilication after 2 weeks. 

Production of H.S: Negative. 

Antagonistic properties: Positive. 

Source: Streptothricosis of liver. 

temarks: Ettlinger ef al. (1958) place 
this species in the S. griseus series. 

Type culture: IMRU 3332. 


251. Streptomyces xanthophaeus Linden- 


291 


ybein, 1952 (Lindenbein, W. Arch. Mikro- 
biol. 17: 361-383, 1952). 

Morphology: No description. 

Glycerol nitrate agar: Growth brownish. 
Aerial mycelium white-gray or reddish-gray. 
Soluble pigment yellow-brown. 

Glycerol malate Growth deep 
orange. Aerial mycelium white-gray to red- 
gray. Soluble pigment deep orange. 

Glucose-asparagine agar: Growth diffuse, 
light yellow. Aerial mycelium white. Solu- 
ble pigment light yellow. 

Nutrient agar: Growth hight brown. Aerial 
mycelium ash-gray to white. Soluble pig- 
vellow-brown. Melanin- 


agar: 


ment yellow to 
negative. 

Glucose-peptone agar: Growth light yel- 
low. Aerial mycelium ash-gray. Soluble pig- 
ment yellow. 

Starch agar: Growth Aerial 
mycelium violet-gray. Hydrolysis rapid. 

Potato: Growth lichenoid. Aerial myce- 
lium gray. No soluble pigment. 

Gelatin: Growth brown. Aerial mycelium 
ash-gray. Soluble pigment yellow-brown. 
Liquefaction strong. 

Milk: Growth lichenoid. Aerial mycelium 
gray to violet. Soluble pigment dark brown. 


lichenoid. 


Peptonization strong. 

Cellulose: No growth. 

Antagonistic properties: Produces geo- 
mycin, active against gram-negative bac- 
teria. 

Habitat: Limestone deposit in Germany. 

Remarks: Related to S. erythraeus and 
S. erythrochromogenes. Kutzner (1956) stud- 
ied five soil isolates. Four strains did not 
form any spirals; one did. The spores were 
smooth. The soluble pigment on glucose- 
peptone agar was dark brown. He thus con- 
sidered this species as melanin-positive. 

Addendum 

After the text of this volume was com- 
pleted, the following newly described forms 
appeared in print: 

Streptomyces aerocolonigenes Shinobu and 


292 


Kawato (Botan. Mag. Tokyo 73: 212-216, 
1960). 

Actinomyces aureoverticillatus KrassiInikov 
and Dzi-Shen (Mikrobiologiya 29: 482-489, 
1960). 

Streptomyces herbaricolor Kawato and 
Shinobu (Mem. Osaka Univ. B 8: 114-119, 
1959). 


THE ACTINOMYCETES, Vol. II 


Streptomyces massasporeus Shinobu and 
Kawato (Botan. Mag. Tokyo 72: 853-854, 
1959). 

Streptomyces — ostreogriseus (Antibiotic 
E-129) Brit. Pat. 799,053, July 30, 1958. 

Streptomyces psammoticus Virgilio and 
Hengeller (Farmaco, Ed. Sci. 15: 164-174, 
1960). 


Chapter 9 


The Genus Micromonospora 


The genus J/icromonospora is character- 
ized by the production in nutrient media of a 
well developed substrate mycelium, 0.2 to 
0.6 w in diameter, partly penetrating into 
the medium. The substrate or vegetative 
hyphae are straight or curved, branching, 
without cross walls. Aerial mycelium is not 
formed at all or only in a rudimentary, non- 
sporulating form, when the hyphae arise 
upward directly from the substrate myce- 
hum. 

Multiplication occurs by means of frag- 
ments of mycelium and special spores formed 
singly. A swelling takes place at the end of 
the sporophore; later the swelling is sepa- 
rated by a cross wall, giving rise to spherical, 
oval, or oblong spores, 1.0 to 1.5 by 0.8 to 
1.2 uw. The sporophores are often branched, 
each branch forming a spore at the end, 
giving rise to a grape-like bunch of spores. 
These germinate in a manner similar to the 
spores of Streptomyces. The mycelium and 
spores are gram-positive, not acid-fast (Fig. 
52). 

The colonies of J/icromonospora are simi- 
lar to those of Streptomyces. They are com- 
pact, leathery, smooth or lichenoid, raised or 
flat. They are frequently colored red or 
orange or yellow, occasionally brown or green 
to almost black or blue. The pigments, ex- 
cept the dark brown, are not dissolved into 
the medium. 

In characterizing species of J/icromono- 
spora, T’ao Ho and Potter (1960) considered 
morphological properties as primary criteria 


for identification of the organisms. The 


293 


important physiological characteristics in- 
cluded the disintegration of cellulose, in- 
version of sucrose, and the reduction of 
nitrate. The investigators emphasized that 
the color of the growth and the form of the 
colony could not serve as basic characteris- 
Certain may different 
colors for the mass of growth and for the 


tics. strains show 
spores. Reproducibility of colony color for a 
given organism could not be obtained on the 
same medium. The color itself was not con- 
sistent, varying through every shade of yel- 
low, orange, pink, red, and brown. Many 
species gave more than one colonial form. 
The large spores of JZ. globosa were very 
helpful in differentiating it from J/. fusca or 
AM. chalcea. 

Micromonospora species are aerobic or 
anaerobic, and mesophilic. They grow 
readily at 25-40°C. Thermal death point of 
the mycelium is 70°C in 2 to 5 minutes; 
spores resist 80°C for 1 to 5 minutes. They 
utilize various carbon and nitrogen sources, 
both organic and inorganic (Fig. 53). 

The type species is icromonospora chalcea 
(Foulerton) Orskoy. 

The genus MJicromonospora comprises nine 
species, which can be classified as follows: 


Classification of the genus Micromonospora 


A. Aerobic. 
I. Sporophores long. 
1. Sporophores showing little branching. 
a. No aerial mycelium. 
2. Micromonospora chalcea 
b. Rudimentary aerial mycelium. 
6. Micromonospora gallica 


294 


2. Sporophores form extensive branching. 
a. Growth colorless; brown spores 
appear in mass. 

7. Micromonospora globosa 

b. Growth pigmented. 
al. Growth green to dark green. 

a2. Spores blue. 
3. Micromonospora coerulea 
b?. Spores black or brown. 
1. Micromonospora bicolor 
b!. Growth pink to orange-colored. 
a2. Pigment not excreted into 
substrate. 
8. Micromonospora parva 
b?. Red-brown pigment excreted 
into substrate. 
5. Micromonospora fusca 
IL. Sporophores short. 
1. Growth brown; spores dark brown. 
4. Micromonospora elongata 

B. Anaerobic. 

9. Micromonospora pro- 


pronica 


Various other micromonosporas have been 
observed in natural substrates, but either 
have not been isolated or only insufficiently 


Ld ‘ 


* 


FIGURE 52. Micromonospora (clumps of spores) 
growing in a compost. 


THE ACTINOMYCETES, Vol. II 


te 
af 
re ~ F 


e \ 


53. Micromonospora (double spores) 


FIGURE 
growing in a compost. 


studied. This is true, for example, of the 
cellulose-decomposing, facultative anaerobic 
form studied by Meyer, 1934 (Prevot, 1955); 
and of J. cabaelli Maquer and Comby 
(Prevot, 1955). It is also true of some of the 
forms reported by Waksman et al. (1939). 
Some of the micromonosporas (J/. mono- 
spora and AM. vulgaris) have